DNA vaccine formulations

ABSTRACT

This invention relates to novel methods and formulations of nucleic acid pharmaceutical products, specifically formulations of nucleic acid vaccine products and nucleic acid gene therapy products. The formulations of the disclosure stabilize the conformation of DNA pharmaceutical products.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. ProvisionalApplication Serial No. 60/017,049, filed Apr. 26, 1996.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

[0002] Not Applicable

REFERENCE TO MICROFICHE APPENDIX

[0003] Not Applicable

FIELD OF THE INVENTION

[0004] The present invention relates to novel formulations of nucleicacid pharmaceutical products, specifically formulations of nucleic acidvaccine products and nucleic acid gene therapy products. Theformulations of the disclosure stabilize the conformation of DNApharmaceutical products. The vaccines, when introduced directly intomuscle cells, induce the production of immune responses whichspecifically recognize human influenza virus.

BACKGROUND OF THE DISCLOSURE

[0005] This invention relates to novel formulations of nucleic acidpharmaceutical products, specifically formulations of nucleic acidvaccine products and nucleic acid gene therapy products. Theformulations of the disclosure stabilize the conformation of DNApharmaceutical products. The vaccines, when introduced directly intomuscle cells, induce the production of immune responses whichspecifically recognize human influenza virus.

[0006] During storage as a pharmaceutical entity, DNA plasmid vaccinesundergo a physicochemical change in which the supercoiled plasmidconverts to the open circular and linear form. A variety of storageconditions (low pH, high temperature, low ionic strength) can acceleratethis process. In this invention, the removal and/or chelation of tracemetal ions (with succinic or malic acid, or with chelators containingmultiple phosphate ligands) from the DNA plasmid solution, from theformulation buffers or from the vials and closures, stabilizes the DNAplasmid from this degradation pathway during storage. In addition,non-reducing free radical scavengers are required to prevent damage ofthe DNA plasmid from free radical production that may still occur, evenin apparently demetalated solutions. Furthermore, the buffer type, pH,salt concentration, light exposure, as well as the type of sterilizationprocess used to prepare the vials, all must be controlled in theformulation to optimize the stability of the DNA vaccine. Lyophilizationof the DNA vaccine in the presence of the appropriate formulationexcipients can also be performed to stabilize the plasmid duringstorage.

[0007] From the scientific literature, the chain scission reactioncausing conversion of supercoiled to open circular to linear DNA plasmidwould be expected to occur via two different chemical mechanisms (sincethese preparations of highly purified DNA do not contain nucleases): (1)depurination followed by β-elimination and/or (2) free radicaloxidation. Although removal of trace metal ions would be expected tosuppress the free radical oxidation mechanism of DNA chain scission,surprisingly, our results indicate that the removal or chelation oftrace metal ions from the DNA containing solution, stabilizes the DNAagainst both mechanisms of degradation, as judged by comparison of ourstability data with the published rates of depurination andβ-elimination (see Lindahl et al., 1972, Biochemistry 19: 3610-3618;Lindahl et al., 1972, Biochemistry 19: 3618-3623). Based on these andother published reports, the removal of trace metal ions would not beexpected to have a significant effect on the rates of depurination orβ-elimination. Therefore, the increase in DNA stability resulting fromthe removal of trace metal ions is much larger than expected, and cannotbe explained on the basis of the published rate constants fordepurination and β-elimination.

[0008] In addition, our data indicates that specific chelating agentssuch as inositol hexaphosphate, tripolyphosphate, succinic and malicacid, increase the stability of plasmid DNA in storage, while othercommonly used chelating agents such as EDTA, desferal,ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) anddiethylenetriaminepenta-acetic acid (DTPA) provide no significantenhancement of stability. These results also suggest that any chelatingagent with multiple phosphate ligands (for example, polyphosphoric acid)will enhance DNA stability. It is not clear from the publishedliterature, however, why inositol hexaphosphate stabilizes DNA, butEDDHA, desferal and DTPA do not. Since the published literature suggeststhat all four of these chelators inhibit the production of hydroxylradicals catalyzed by iron, it was expected that all of these reagentswould provide enhanced DNA stability (by chelating trace metal ions andinhibiting the production of free radicals), but this was not observed.Moreover, the literature reports that both EDTA and ATP support metalion catalyzed hydroxyl radical production, but we have observed thattripolyphosphate (the metal binding moiety of ATP) enhances DNAstability while EDTA does not. Therefore, the protective effects of themetal ion chelators do not appear to be directly correlated with theirability to support the production of hydroxyl radicals. Theidentification of the appropriate chelators to stabilize DNAformulations will require empirical testing as described in this work.

[0009] In addition to the removal and/or chelation of trace metal ions,the use of non-reducing free radical scavengers is important forstabilizing DNA formulations during storage. Our results indicate thatethanol, methionine, glycerol and dimethyl sulfoxide enhance DNAstability, suggesting that their protective effect is due to thescavenging of free radicals. Furthermore, our results indicate thatscavengers capable of serving as reducing agents, such as ascorbic acid,greatly accelerate DNA degradation, presumably by acting as a reducingagent to keep trace metal ions in their reduced (most damaging) state.Our results also indicate that several scavengers expected to stabilizeDNA (based on known rate constants with hydroxyl radical) unexpectedlyaccelerated DNA degradation, or provided no increase in stability. Forexample, pentoxifylline and para-aminobenzoic acid are hydroxyl radicalscavengers with large rate constants for hydroxyl radicals (k=1.1×10¹⁰M⁻¹ s⁻¹; see Freitas and Filipe, 1995, Biol. Trace Elem. Res. 47:307-311; Hu et al., 1995, J. Nutr. Biochem. 6: 504-508), yetpentoxifylline did not enhance stability and p-aminobenzoic acidactually accelerated DNA degradation. Because of these results, theempirical screening of a number of free radical scavengers has been themost effective means of identifying useful compounds.

[0010] To maximize DNA stability in a pharmaceutical formulation, thetype of buffer, salt concentration, pH, light exposure as well as thetype of sterilization process used to prepare the vials are allimportant parameters that must be controlled in the formulation tofurther optimize the stability. Furthermore, lyophilization of the DNAvaccine with appropriate formulation excipients can also be performedenhance DNA stability, presumably by reducing molecular motion viadehydration. Therefore, our data suggest that the formulation that willprovide the highest stability of the DNA vaccine will be one thatincludes a demetalated solution containing a buffer (phosphate orbicarbonate) with a pH in the range of 7-8, a salt (NaCl, KCl or LiCl)in the range of 100-200 mM, a metal ion chelator (succinate, malate,inositol hexaphosphate, tripolyphosphate or polyphosphoric acid), anon-reducing free radical scavenger (ethanol, glycerol, methionine ordimethyl sulfoxide) and the highest appropriate DNA concentration in asterile glass vial, packaged to protect the highly purified, nucleasefree DNA from light.

[0011] The instant formulations and methods are exemplified with a DNAvaccine against influenza. Nothing in this disclosure should beconstrued as limiting the formulations and methods to the specific DNAvaccine.

[0012] Influenza is an acute febrile illness caused by infection of therespiratory tract with influenza A or B virus. Outbreaks of influenzaoccur worldwide nearly every year with periodic epidemics or pandemics.Influenza can cause significant systemic symptoms, severe illness (suchas viral pneumonia) requiring hospitalization, and complications such assecondary bacterial pneumonia. Recent U.S. epidemics are thought to haveresulted in >10,000 (up to 40,000) excess deaths per year and5,000-10,000 deaths per year in non-epidemic years. The best strategyfor prevention of the morbidity and mortality associated with influenzais vaccination. The current licensed vaccines are derived from virusgrown in eggs, then inactivated, and include three virus strains (two Astrains and one B strain). Three types of vaccines are available:whole-virus, subvirion, and purified surface antigen. Only the lattertwo are used in children because of increased febrile responses with thewhole-virus vaccine. Children under the age of 9 require twoimmunizations, while adults require only a single injection. However, ithas been suggested [see Medical Letter 32:89-90, Sep. 17, 1993] that“patients vaccinated early in the autumn might benefit from a seconddose in the winter or early spring,” due to the observations that insome elderly patients, the antibody titers following vaccination maydecline to less-than-protective levels within four months or less. Thesevaccines are reformulated every year by predicting which recent viralstrains will clinically circulate and evaluating which new virulentstrain is expected to be predominant in the coming flu season.Revaccination is recommended annually.

[0013] The limitations of the licensed vaccine include the following:

[0014] 1) Antigenic variation, particularly in A strains of influenza,results in viruses that are not neutralized by antibodies generated by aprevious vaccine (or previous infection). New strains arise by pointmutations (antigenic drift) and by reassortment (antigenic shift) of thegenes encoding the surface glycoproteins (hemagglutinin [HA] andneuraminidase), while the internal proteins are highly conserved amongdrifted and shifted strains. Immunization elicits “homologous”strain-specific antibody-mediated immunity, not “heterologous”group-common immunity based on cell-mediated immunity.

[0015] 2) Even if the predominant, circulating strains of influenzavirus do not shift or drift significantly from one year to the next,immunization must be given each year because antibody titers decline.Although hemagglutination-inhibiting (HI) and neutralizing antibodiesare reported by some to persist for months to years with a subsequentgradual decline, the Advisory Committee on Immunization Practices citesthe decline in antibody titers in the year following vaccination as areason for annual immunization even when there has been no major driftor shift. (HI antibodies inhibit the ability of influenza virus toagglutinate red blood cells. Like neutralizing antibodies, they areprimarily directed against the HA antigen.

[0016] Hemagglutination inhibition tests are easier and less expensiveto perform than neutralization assays are, and thus are often used as ameans to assess the ability of antibodies raised against one strains ofinfluenza to react to a different strain). As mentioned above, TheMedical Letter suggests that certain high-risk, older individuals shouldbe vaccinated twice in one season due to short-lived protective antibodytiters.

[0017] 3) The effectiveness of the vaccine is suboptimal. Development ofthe next season's vaccine relies upon predicting the upcomingcirculating strains (via sentinel sampling in Asia), which is inexactand can result in a poor match between strains used for the vaccine andthose that actually circulate in the field. Moreover, as occurred duringthe 1992-1993 flu season, a new H3N2 strain (A/Beijing/92) becameclinically apparent during the latter phase of the flu season. Thisprompted a change in the composition of the 1993-1994 vaccine, due topoor cross-reactivity with A/Beijing/92 of the antibody induced by theearlier H3N2 strain (A/Beijing/89) due to antigenic shift. However, dueto the length of time needed to make and formulate the current licensedvaccine, the new vaccine strain could not be introduced during the1992-1993 season despite the evidence for poor protection from theexisting vaccine and the increased virulence of the new circulating H3N2strain.

[0018] Characteristics of an Ideal Universal Influenza Vaccine includethe following:

[0019] 1) Generation of group-common (heterologous) protection.

[0020] 2) Increased breadth of antibody response. Because CTL arethought to play a role in recovery from disease, a vaccine based solelyupon a CTL response would be expected to shorten the duration of illness(potentially to the point of rendering illness subclinical), but itwould not prevent illness completely.

[0021] 3) Increased duration of antibody responses. Because one of thevery groups that is at highest risk for the morbidity and mortality ofinfluenza infection (elderly) is also the group in whom protectiveantibody titers may decline too rapidly for annual immunization to beeffective, an improved vaccine should generate protective titers ofantibody that persist longer.

[0022] Intramuscular inoculation of polynucleotide constructs, i.e., DNAplasmids encoding proteins have been shown to result in the in situgeneration of the protein in muscle cells. By using cDNA plasmidsencoding viral proteins, both antibody and CTL responses were generated,providing homologous and heterologous protection against subsequentchallenge with either the homologous or cross-strain protection,respectively. Each of these types of immune responses offers a potentialadvantage over existing vaccination strategies. The use of PNVs(polynucletide vaccines) to generate antibodies may result in anincreased duration of the antibody responses as well as the provision ofan antigen that can have both the exact sequence of the clinicallycirculating strain of virus as well as the proper post- translationalmodifications and conformation of the native protein (vs. a recombinantprotein). The generation of CTL responses by this means offers thebenefits of cross-strain protection without the use of a livepotentially pathogenic vector or attenuated virus.

[0023] Therefore, this invention contemplates any of the known methodsfor introducing nucleic acids into living tissue to induce expression ofproteins. This invention provides a method for introducing viralproteins into the antigen processing pathway to generate virus-specificCTLs. Thus, the need for specific therapeutic agents capable ofeliciting desired prophylactic immune responses against viral pathogensis met for influenza virus by this invention. Of particular importancein this therapeutic approach is the ability to induce T-cell immuneresponses which can prevent infections even of virus strains which areheterologous to the strain from which the antigen gene was obtained.Therefore, this invention provides DNA constructs encoding viralproteins of the human influenza virus nucleoprotein (NP), hemagglutinin(HA), neuraminidase (NM), matrix (M), nonstructural (NS), polymerase(PB1 and PB2=basic polymerases 1 and 2; PA=acidic polymerase) or any ofthe other influenza genes which encode products which generate specificCTLs.

SUMMARY OF THE INVENTION

[0024] This invention relates to novel formulations of nucleic acidpharmaceutical products, specifically formulations of nucleic acidvaccine products and nucleic acid gene therapy products. Theformulations of the disclosure stabilize the conformation of DNApharmaceutical products. The vaccines, when introduced directly intomuscle cells, induce the production of immune responses whichspecifically recognize human influenza virus.

[0025] During storage as a pharmaceutical entity, DNA plasmid vaccinesundergo a physicochemical change in which the supercoiled plasmidconverts to the open circular and linear form. A variety of storageconditions (low pH, high temperature, low ionic strength) can acceleratethis process. In this invention, the removal and/or chelation of tracemetal ions (with succinic or malic acid, or with chelators containingmultiple phosphate ligands) from the DNA plasmid solution, from theformulation buffers or from the vials and closures, stabilizes the DNAplasmid from this degradation pathway during storage. In addition,non-reducing free radical scavengers are required to prevent damage ofthe DNA plasmid from free radical production that may still occur, evenin apparently demetalated solutions. Furthermore, the buffer type, pH,salt concentration, light exposure, as well as the type of sterilizationprocess used to prepare the vials, all must be controlled in theformulation to optimize the stability of the DNA vaccine. Lyophilizationof the DNA vaccine in the presence of the appropriate formulationexcipients can also be performed to stabilize the plasmid duringstorage.

[0026] From the scientific literature, the chain scission reactioncausing conversion of supercoiled to open circular to linear DNA plasmidwould be expected to occur via two different chemical mechanisms (sincethese preparations of highly purified DNA do not contain nucleases): (1)depurination followed by β-elimination and/or (2) free radicaloxidation. Although removal of trace metal ions would be expected tosuppress the free radical oxidation mechanism of DNA chain scission,surprisingly, our results indicate that the removal or chelation oftrace metal ions from the DNA containing solution, stabilizes the DNAagainst both mechanisms of degradation, as judged by comparison of ourstability data with the published rates of depurination andβ-elimination (see Lindahl et al., 1972, Biochemistry 19: 3610-3618;Lindahl et al., 1972, Biochemistry 19: 3619-3623). Based on these andother published reports, the removal of trace metal ions would not beexpected to have a significant effect on the rates of depurination orβ-elimination. Therefore, the increase in DNA stability resulting fromthe removal of trace metal ions is much larger than expected, and cannotbe explained on the basis of the published rate constants fordepurination and β-elimination.

[0027] In addition, our data indicates that specific chelating agentssuch as inositol hexaphosphate, tripolyphosphate, succinic and malicacid, increase the stability of plasmid DNA in storage, while othercommonly used chelating agents such as EDTA, desferal,ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) anddiethylenetriaminepenta-acetic acid (DTPA) provide no significantenhancement of stability. These results also suggest that any chelatingagent with multiple phosphate ligands (for example, polyphosphoric acid)will enhance DNA stability. It is not clear from the publishedliterature, however, why inositol hexaphosphate stabilizes DNA, butEDDHA, desferal and DTPA do not. Since the published literature suggeststhat all four of these chelators inhibit the production of hydroxylradicals catalyzed by iron, it was expected that all of these reagentswould provide enhanced DNA stability (by chelating trace metal ions andinhibiting the production of free radicals), but this was not observed.Moreover, the literature reports that both EDTA and ATP support metalion catalyzed hydroxyl radical production, but we have observed thattripolyphosphate (the metal binding moiety of ATP) enhances DNAstability while EDTA does not. Therefore, the protective effects of themetal ion chelators do not appear to be directly correlated with theirability to support the production of hydroxyl radicals. Theidentification of the appropriate chelators to stabilize DNAformulations will require empirical testing as described in this work.

[0028] In addition to the removal and/or chelation of trace metal ions,the use of non-reducing free radical scavengers is important forstabilizing DNA formulations during storage. Our results indicate thatethanol, methionine, glycerol and dimethyl sulfoxide enhance DNAstability, suggesting that their protective effect is due to thescavenging of free radicals. Furthermore, our results indicate thatscavengers capable of serving as reducing agents, such as ascorbic acid,greatly accelerate DNA degradation, presumably by acting as a reducingagent to keep trace metal ions in their reduced (most damaging ) state.Our results also indicate that several scavengers expected to stabilizeDNA (based on known rate constants with hydroxyl radical) unexpectedlyaccelerated DNA degradation, or provided no increase in stability. Forexample, pentoxifylline and para-aminobenzoic acid are hydroxyl radicalscavengers with large rate constants for hydroxyl radicals (k=1.1×10¹⁰M⁻¹ s⁻¹; see Freitas and Filipe, 1995, Biol. Trace Elem. Res. 47:307-311; Hu et al., 1995, J. Nutr. Biochem. 6: 504-508), yetpentoxifylline did not enhance stability and p-aminobenzoic acidactually accelerated DNA degradation. Because of these results, theempirical screening of a number of free radical scavengers has been themost effective means of identifying useful compounds.

[0029] To maximize DNA stability in a pharmaceutical formulation, thetype of buffer, salt concentration, pH, light exposure as well as thetype of sterilization process used to prepare the vials are allimportant parameters that must be controlled in the formulation tofurther optimize the stability. Furthermore, lyophilization of the DNAvaccine with appropriate formulation excipients can also be performedenhance DNA stability, presumably by reducing molecular motion viadehydration. Therefore, our data suggest that the formulation that willprovide the highest stability of the DNA vaccine will be one thatincludes a demetalated solution containing a buffer (phosphate orbicarbonate) with a pH in the range of 7-8, a salt (NaCl, KCl or LiCl)in the range of 100-200 mM, a metal ion chelator (succinate, malate,inositol hexaphosphate, tripolyphosphate or polyphosphoric acid), anon-reducing free radical scavenger (ethanol, glycerol, methionine ordimethyl sulfoxide) and the highest appropriate DNA concentration in asterile glass vial, packaged to protect the highly purified, nucleasefree DNA from light.

[0030] Data is presented in this specification which exemplifies severaladditional DNA vaccine formulations. More specifically, the presentinvention relates to DNA vaccine formulations which comprise ademetalated solution containing a physiologically acceptable bufferwithin a pH range from at least greater than about 8.0 to about at least9.5, a salt (including but not limited to NaCl, KCl or LiCl) in therange of up to about at 300 mM, and the metal ion chelator EDTA (in therange of up to about 5 mM) in combination with the free radicalscavenger ethanol (in the range of up to about 3%) and the highestappropriate DNA concentration in a sterile glass vial, packaged toprotect the highly purified, nuclease free DNA from light and in aphysiologically acceptable buffer.

[0031] In a specific aspect of the present invention, the DNA vaccineformulations comprise a combination of EDTA and ethanol, NaCl at aconcentration from about 100 mM to about 200 mM, EDTA in the range fromabout 1 μM to about 1 mM, ethanol present up to about 2%, all in thehighest appropriate DNA concentration in a sterile glass vial, packagedto protect the highly purified, nuclease free DNA from light and in aphysiologically acceptable buffer.

[0032] In another embodiment of the DNA vaccine formulations comprisinga combination of EDTA and ethanol, NaCl is present from about 100 mM toabout 200 mM, EDTA is present from about 1 μM to about 750 μM, ethanolis present from about 0.5% to about 2.5%, all in the highest appropriateDNA concentration in a sterile glass vial, packaged to protect thehighly purified, nuclease free DNA from light and in a physiologicallyacceptable buffer which preferably is Tris-HCl at a pH from about 8.0 toabout 9.0 and glycine from about pH 9.0 to about pH 9.5. It will beunderstood that other known buffers with a buffering capacity withinvarious pH ranges, such as pH 8.0 to 9.5, may be utilized in the variousDNA vaccine formulations of the present invention.

[0033] In an especially preferred aspect of the present inventionwherein the DNA vaccine formulations comprise a combination of EDTA andethanol, NaCl at a concentration from about 100 mM to about 200 mM, EDTAis present at about 500 μM, ethanol is present at about 1.0%, all in thehighest appropriate DNA concentration in a sterile glass vial, packagedto protect the highly purified, nuclease free DNA from light and in aphysiologically acceptable buffer which preferably is Tris-HCl at a pHfrom about 8.5 to about 9.0.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D show the effect of containertype on supercoil content of Influenza DNA vaccine, HA (Georgia/93)during storage. DNA plasmid solution was prepared at 100 mcg/mL DNA insaline. Supercoil content of plasmid was determined by agarose gelelectrophoresis. Panel A-glass; Panel B-siliconized glass; PanelC-autoclaved plastic; Panel D-gamma-plastic.

[0035]FIG. 2 shows the effect of DNA plasmid concentration on supercoilcontent of Influenza DNA vaccine, HA (Georgia/93) during storage at 37°C. DNA plasmid solution was prepared at 20-1800 mcg/mL. Supercoilcontent of plasmid was determined by agarose gel electrophoresis.

[0036]FIG. 3 shows the UV-melting curve of plasmid DNA stored in 10 mMphosphate buffer (pH 6.0) over the NaCl concentration range of 0-200 mM.Samples contained 100 mcg/ml plasmid DNA with the followingconcentrations of NaCl; (a) 0 mM, (b) 5 mM, (c) 20 mM, (d) 50 mM, (e)100 mM, (f) 200 mM.

[0037]FIG. 4 shows the stability of plasmid DNA in the presence ofincreasing concentrations of NaCl. Samples were incubated at 60° C. for48 hours then analyzed by agarose gel electrophoresis. At time zerosamples contained 90% supercoiled DNA.

[0038]FIG. 5A, FIG. 5B and FIG. 5C show the stability of plasmid DNA inthe presence of divalent cations in TE (Panel A), PBS (Panel B) andsaline (Panel C). Samples were heated at 60° C. for 48 hours at aconcentration of 100 mcg/mL then analyzed for degradation by agarose gelelectrophoresis. At time zero, samples contained 90% supercoiled DNA.

[0039]FIG. 6 shows the UV-melting curve of DNA over the pH range 4.5-10.The samples contained 20 mcg/mL plasmid in the following pH buffers: (a)citrate pH 4.5, (b) phosphate pH 6.0, (c) phosphate pH 6.5, (d)phosphate pH 7.0, (e) phosphate pH 7.5, (f) phosphate pH 8.0, (g) boratepH 10.

[0040]FIG. 7A shows the conformational stability of plasmid DNA in PBSand TE (10 mM Tris-Cl, 1 mM EDTA) buffered over the pH range 6.0 to 8.5.Samples were incubated at 100 mcg/mL for 48 hours at 60° C. thenanalyzed for degradation by electrophoresis. At time zero samplescontained 90% supercoiled DNA.

[0041]FIG. 7B shows the effect of buffer type and solution pH on thesupercoil content (DNA stability) of Influenza DNA vaccine duringstorage at 37° C. DNA plasmid solutions were prepared at 20 mcg/mL. DNAwas formulated in saline alone, or in saline with a Tris, Hepes,Phosphate or Sodium Bicarbonate buffer. Buffer pH was measured at timezero (room temperature) and at 3 months (37° C.). Supercoil content ofplasmid was determined by agarose gel electrophoresis.

[0042]FIG. 8A and FIG. 8B show the effect of PBS vs. saline (0.9% w/vNaCl) formulation on the supercoil content of Influenza DNA(HA-Georgia/93) vaccine during storage at 25° C. (Panel B) and 37° C.(Panel A). DNA plasmid solutions were prepared at 2 mcg/mL. Supercoilcontent of plasmid was determined by agarose gel electrophoresis.

[0043]FIG. 9 shows an induced mouse immune response as measured by HItiter for a single injection of PBS vs. saline formulated Influenza DNAvaccine, HA (Georgia/93). DNA plasmid solutions were prepared at variousdoses and 200 microliters of the vaccine was injected in each mouse (tenmice per dose per formulation).

[0044]FIG. 10 shows the effect of exposure to light on the supercoilcontent of Influenza DNA vaccine during storage at 25° C. DNA plasmidsolutions were prepared at 100 mcg/mL DNA in saline. Supercoil contentof plasmid was determined by agarose gel electrophoresis.

[0045]FIG. 11 shows the effect of free radical scavengers on thesupercoil content of Influenza DNA vaccine during storage at 37° C. DNAplasmid solutions were prepared at 100 mcg/mL DNA in saline. Supercoilcontent of plasmid was determined by agarose gel electrophoresis.

[0046]FIG. 12 shows the effect of lyophilization on the supercoilcontent of Influenza DNA vaccine during storage at 37° C. DNA plasmidsolutions were prepared at 20 mcg/mL DNA in either phosphate buffer orphosphate buffered saline (pH 7) containing the indicated sugars.Approximately 0.7 mL of the formulated DNA solution was placed in 3 mLglass vials and then lyophilized. Freeze dried DNA formulations at Day 0showed no change in plasmid supercoil content compared to liquidcontrols (no lyophilization). Supercoil content of plasmid wasdetermined by agarose gel electrophoresis. A-PBS (liquid control; nolyophilization); B-PBS containing 5% mannitol, lyophilized; C-Phosphatebuffer containing 5% mannitol, lyophilized; D-PBS containing 5% lactose,lyophilized; E-Phosphate buffer containing 5% sucrose, lyophilized;F-Phosphate buffer containing 4% mannitol and 1% lactose, lyophilized;G-Phosphate buffer containing 4% mannitol and 1% sucrose, lyophilized.The formulations were stored for 1 month at 37° C.

[0047]FIG. 13 shows the effect of pH on DNA stability (as a percentageof initial supercoiled plasmid DNA remaining) after two weeks at 50° C.in 20 mM Bis-Tris-Propane, 150 mM NaCl. Control is DNA in PBS at pH 7.1.

[0048]FIG. 14 shows the effect of buffer type and pH on DNA stability at50° C. at a DNA concentration of 20 mcg/mL. DNA stability is measured as% of initial supercoiled (SC) DNA remaining.

[0049]FIG. 15 shows the effect of demetalation on DNA stability at 50°C., pH 7.2 in 2 mcg/mL of DNA. DNA stability is measured as % of initialsupercoiled (SC) DNA remaining.

[0050]FIG. 16 shows the effect of demetalation on DNA stability at 50°C., pH 8.0 in 2 mcg/mL of DNA. DNA stability is measured as % of initialsupercoiled (SC) DNA remaining.

[0051]FIG. 17 shows the effect of demetalation on DNA stability informulations containing succinate and ethanol. DNA stability is measuredas % of initial supercoiled (SC) DNA remaining.

[0052]FIG. 18 shows the effect of demetalation on DNA stability informulations containing bicarbonate and borate. DNA stability ismeasured as % of initial supercoiled (SC) DNA remaining.

[0053]FIG. 19 shows the effect of demetalation on DNA stability in PBSand bicarbonate formulations at 30° C. DNA stability is measured as % ofinitial supercoiled (SC) DNA remaining.

[0054]FIG. 20 shows the effect of EDTA and ethanol on DNA stability at50° C. in PBS at pH 7.2. DNA stability is measured as % of initialsupercoiled (SC) DNA remaining.

[0055]FIG. 21 shows the effect of EDTA and ethanol on DNA stability at50° C. in PBS at pH 8.0. DNA stability is measured as % of initialsupercoiled (SC) DNA remaining.

[0056]FIG. 22 shows the effect of iron on DNA stability in formulationscontaining EDTA/ethanol at 50° C. DNA stability is measured as % ofinitial supercoiled (SC) DNA remaining.

[0057]FIG. 23 shows the effect of metal iron chelators on DNA stabilityin PBS at pH 8.0. DNA stability is measured as % of initial supercoiled(SC) DNA remaining.

[0058]FIG. 24 shows the effect of metal ion chelators on DNA stabilityin PBS at pH 8.0 in the presence of ethanol. DNA stability is measuredas % of initial supercoiled (SC) DNA remaining.

[0059]FIG. 25 shows the effect of EDTA concentration on DNA stability inPBS at 1% ethanol. DNA stability is measured as % of initial supercoiled(SC) DNA remaining.

[0060]FIG. 26 shows the stability of lyophilized (formulations #1-6) andliquid (formulations 7-9) DNA formulations after 4 months at 50° C.Formulation #1 is 5% sucrose, 5 mM NaPO₄; formulation #2 is 5% sucrose,5 mM NaPO₄, demetalated; formulation #2 is 5% sucrose, 5 mM NaPO₄, 150mM NaCl; formulation #4 is 5% sucrose, 5 mM NaPO₄, 150 mM NaCl,demetalated; formulation #5 is 4% mannose, 1% sucrose, 5 mM NaPO₄;formulation #6 is 4% mannose, 1% sucrose, 5 mM NaPO₄, demetalated;formulation #7 is 10 mM NaPO₄, 150 mM NaCl, 0.5 mM EDTA and 2% ethanolat pH 8.0; formulation #8 is 20 mM Tris, 150 NaCl, 10 mM succinate, 2%ethanol at pH 8.2; and formulation #9 is 20 mM glycine, 150 mM NaCl, 10mM succinate , 2% ethanol at pH 9.0. DNA stability is measured as % ofinitial supercoiled (SC) DNA remaining.

[0061]FIG. 27 shows the effect of light on DNA stability in formulationscontaining iron and EDTA. DNA stability is measured as % of initialsupercoiled (SC) DNA remaining.

[0062]FIG. 28 shows the effect of light on DNA stability in formulationscontaining iron, EDTA and ethanol. DNA stability is measured as % ofinitial supercoiled (SC) DNA remaining.

[0063]FIG. 29 shows an Arrhenius plot of depurination in PBS, 1%ethanol, and 0.5 mM EDTA at pH 7.4.

[0064]FIG. 30 shows an Arrhenius plot of β-elimination in PBS, 1%ethanol, and 0.5 mM EDTA at pH 7.4.

[0065]FIG. 31 shows a prediction of DNA stability at 50° C. usingpublished and measured values of k₁ and k₂.

[0066]FIG. 32 shows a prediction of DNA stability at 30° C. based onmeasured values of k₁ and k₂ at pH 7.4.

DETAILED DESCRIPTION OF THE INVENTION

[0067] This invention relates to novel formulations of nucleic acidpharmaceutical products, specifically formulations of nucleic acidvaccine products and nucleic acid gene therapy products. Theformulations of the disclosure stabilize the conformation of DNApharmaceutical products. The vaccines, when introduced directly intomuscle cells, induce the production of immune responses whichspecifically recognize human influenza virus.

[0068] During storage as a pharmaceutical entity, DNA plasmid vaccinesundergo a physicochemical change in which the supercoiled plasmidconverts to the open circular and linear form. A variety of storageconditions (low pH, high temperature, low ionic strength) can acceleratethis process. In this invention, the removal and/or chelation of tracemetal ions (with succinic or malic acid, or with chelators containingmultiple phosphate ligands) from the DNA plasmid solution, from theformulation buffers or from the vials and closures, stabilizes the DNAplasmid from this degradation pathway during storage. In addition,non-reducing free radical scavengers are required to prevent damage ofthe DNA plasmid from free radical production that may still occur, evenin apparently demetalated solutions. Furthermore, the buffer type, pH,salt concentration, light exposure, as well as the type of sterilizationprocess used to prepare the vials, all must be controlled in theformulation to optimize the stability of the DNA vaccine. Lyophilizationof the DNA vaccine in the presence of the appropriate formulationexcipients can also be performed to stabilize the plasmid duringstorage.

[0069] From the scientific literature, the chain scission reactioncausing conversion of supercoiled to open circular to linear DNA plasmidwould be expected to occur via two different chemical mechanisms (sincethese preparations of highly purified DNA do not contain nucleases): (1)depurination followed by β-elimination and/or (2) free radicaloxidation. Although removal of trace metal ions would be expected tosuppress the free radical oxidation mechanism of DNA chain scission,surprisingly, our results indicate that the removal or chelation oftrace metal ions from the DNA containing solution, stabilizes the DNAagainst both mechanisms of degradation, as judged by comparison of ourstability data with the published rates of depurination andβ-elimination (see Lindahl et al., Biochemistry 19: 3610-3618, 1972;Lindahl et al., Biochemistry 19: 3618-3623, 1972). Based on these andother published reports, the removal of trace metal ions would not beexpected to have a significant effect on the rates of depurination orβ-elimination. Therefore, the increase in DNA stability resulting fromthe removal of trace metal ions is much larger than expected, and cannotbe explained on the basis of the published rate constants fordepurination and β-elimination.

[0070] In addition, our data indicates that specific chelating agentssuch as inositol hexaphosphate, tripolyphosphate, succinic and malicacid, increase the stability of plasmid DNA in storage, while othercommonly used chelating agents such as EDTA, desferal,ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) anddiethylenetriaminepenta-acetic acid (DTPA) provide no significantenhancement of stability. These results also suggest that any chelatingagent with multiple phosphate ligands (for example, polyphosphoric acid)will enhance DNA stability. It is not clear from the publishedliterature, however, why inositol hexaphosphate stabilizes DNA, butEDDHA, desferal and DTPA do not. Since the published literature suggeststhat all four of these chelators inhibit the production of hydroxylradicals catalyzed by iron, it was expected that all of these reagentswould provide enhanced DNA stability (by chelating trace metal ions andinhibiting the production of free radicals), but this was not observed.Moreover, the literature reports that both EDTA and ATP support metalion catalyzed hydroxyl radical production, but we have observed thattripolyphosphate (the metal binding moiety of ATP) enhances DNAstability while EDTA does not. Therefore, the protective effects of themetal ion chelators do not appear to be directly correlated with theirability to support the production of hydroxyl radicals. Theidentification of the appropriate chelators to stabilize DNAformulations will require empirical testing as described in this work.

[0071] In addition to the removal and/or chelation of trace metal ions,the use of non-reducing free radical scavengers is important forstabilizing DNA formulations during storage. Our results indicate thatethanol, methionine, glycerol and dimethyl sulfoxide enhance DNAstability, suggesting that their protective effect is due to thescavenging of free radicals. Furthermore, our results indicate thatscavengers capable of serving as reducing agents, such as ascorbic acid,greatly accelerate DNA degradation, presumably by acting as a reducingagent to keep trace metal ions in their reduced (most damaging ) state.Our results also indicate that several scavengers expected to stabilizeDNA (based on known rate constants with hydroxyl radical) unexpectedlyaccelerated DNA degradation, or provided no increase in stability. Forexample, pentoxifylline and para-aminobenzoic acid are hydroxyl radicalscavengers with large rate constants for hydroxyl radicals (k=1.1×10¹⁰M⁻¹ s⁻¹; see Freitas and Filipe, Biol. Trace Elem. Res. 47: 307-311,1995; Hu et al., J. Nutr. Biochem. 6: 504-508, 1995), yet pentoxifyllinedid not enhance stability and p-aminobenzoic acid actually acceleratedDNA degradation. Because of these results, the empirical screening of anumber of free radical scavengers has been the most effective means ofidentifying useful compounds.

[0072] To maximize DNA stability in a pharmaceutical formulation, thetype of buffer, salt concentration, pH, light exposure as well as thetype of sterilization process used to prepare the vials are allimportant parameters that must be controlled in the formulation tofurther optimize the stability. Furthermore, lyophilization of the DNAvaccine with appropriate formulation excipients can also be performedenhance DNA stability, presumably by reducing molecular motion viadehydration. Therefore, our data suggest that the formulation that willprovide the highest stability of the DNA vaccine will be one thatincludes a demetalated solution containing a buffer (phosphate orbicarbonate) with a pH in the range of 7-8, a salt (NaCl, KCl or LiCl)in the range of 100-200 mM, a metal ion chelator (succinate, malate,inositol hexaphosphate, tripolyphosphate or polyphosphoric acid), anon-reducing free radical scavenger (ethanol, glycerol, methionine ordimethyl sulfoxide) and the highest appropriate DNA concentration in asterile glass vial, packaged to protect the highly purified, nucleasefree DNA from light.

[0073] DNA constructs encoding influenza viral proteins elicitprotective immune responses in animals. Immune responses in animals haveincluded antibody and CTL generation in mice, antibody generation inferrets and primates, and protection from viral challenge in mice andferrets with homologous, drifted and shifted strains of influenza.Perhaps the most striking result of immunization with DNA encoding viralproteins was the ability to confer protection against distinct subtypesof virus. This suggests that adding a CTL-eliciting component to avaccine should serve to mitigate the impact of new variants which arisein mid-season or are unanticipated when the vaccine strains are choseneach year for the following year. Importantly, immunization with cDNAvectors encoding an HA, NP and M1 gene was able to protect moreeffectively against a drifted strain of virus in ferrets than was thelicensed vaccine. This provides a justification for the use ofconstructs encoding internal genes in the IDV (Influenza DNA vaccine).

[0074] In one embodiment, the vaccine product will consist of separateDNA plasmids encoding, for example, HA from the 3 prevalent clinicalstrains representing A/H1N1 (A/Texas/91), A/H3N2 (A/Georgia/93), and B(B/Panama/90) viruses as well as DNA constructs encoding the internalconserved proteins NP and M1 (matrix) from both A (Beijing/89; H3N2) andB strains in order to provide group-common protection against driftedand shifted antigens. The HA DNA will function by generating HA andresulting neutralizing antibodies against HA. This will betype-specific, with some increased breadth of protection against adrifted strain compared to the current licensed, protein-based vaccine.The NP and M1 constructs will result in the generation of CTL which willprovide cross-strain protection with potentially lower viral loads andwith acceleration of recovery from illness. The expected persistence ofthe DNA constructs (in an episomal, non-replicating, non-integrated formin the muscle cells) is expected to provide an increased duration ofprotection compared to the current vaccine.

[0075] The anticipated advantages over the current, licensed vaccinesinclude: increased breadth of protection due to CTL responses ±increased breadth of antibody, and increased duration of protection. TheIDV approach avoids the need to make, select and propagate reassortantsas is done for the current licensed vaccine since a new DNA constructcan be made more directly from a clinical field isolate. lymphocyteresponse.

[0076] The intramuscular (i.m.). injection of a DNA expression vectorencoding a conserved, internal protein of influenza A resulted in thegeneration of significant protective immunity against subsequent viralchallenge. In particular, NP-specific antibodies and primary CTLs wereproduced. NP DNA immunization resulted in decreased viral lung titers,inhibition of weight loss, and increased survival, compared to controls.The protective immune response was not mediated by the NP-specificantibodies, as demonstrated by the lack of effect of NP antibodies alone(see Example 4) in combating a virus infection, and was thus likely dueto NP-specific cellular immunity. Moreover, significant levels ofprimary CTLs directed against NP were generated. The protection wasagainst a virulent strain of influenza A that was heterologous to thestrain from which the DNA was cloned. Additionally, the challenge strainarose more than three decades after the A/PR/8/34 strain, indicatingthat immune responses directed against conserved proteins can beeffective despite the antigenic shift and drift of the variable envelopeproteins. Because each of the influenza virus gene products exhibit somedegree of conservation, and because CTLs may be generated in response tointracellular expression and MHC processing, it is predictable thatother influenza virus genes will give rise to responses analogous tothat achieved for NP. Methods for identifying immunogenic epitopes arenow well known in the art [see for example Shirai et al.,1992, J.Immunol 148:1657-1667; Choppin et al., 1991, J. Immunol 147:575-583;Calin-Laurens, et al., 1992, Vaccine 11:974-978]. Thus, many of thesegenes have been cloned, as shown by the cloned and sequenced junctionsin the expression vector (see below) such that these constructs areprophylactic agents in available form.

[0077] The standard techniques of molecular biology for preparing andpurifying DNA constructs enable the preparation of the DNA therapeuticsof this invention. While standard techniques of molecular biology aretherefore sufficient for the production of the products of thisinvention, the specific constructs disclosed herein provide noveltherapeutics which surprisingly produce cross-strain protection, aresult heretofore unattainable with standard inactivated whole virus orsubunit protein vaccines.

[0078] The amount of expressible DNA to be introduced to a vaccinerecipient will depend on the strength of the transcriptional andtranslational promoters used in the DNA construct, and on theimmunogenicity of the expressed gene product. In general, animmunologically or prophylactically effective dose of about 1 μg to 1mg, and preferably about 10 μg to 300 μg is administered directly intomuscle tissue. Subcutaneous injection, intradermal introduction,impression through the skin, and other modes of administration such asintraperitoneal, intravenous, or inhalation delivery are alsocontemplated. It is also contemplated that booster vaccinations are tobe provided.

[0079] The DNA may be naked, that is, unassociated with any proteins,adjuvants or other agents which impact on the recipients immune system.In this case, it is desirable for the DNA to be in a physiologicallyacceptable solution, such as, but not limited to, sterile saline orsterile buffered saline. Alternatively, the DNA may be associated withliposomes, such as lecithin liposomes or other liposomes known in theart, as a DNA-liposome mixture, (see for example WO93/24640) or the DNAmay be associated with an adjuvant known in the art to boost immuneresponses, such as a protein or other carrier. Agents which assist inthe cellular uptake of DNA, such as, but not limited to, calcium ions,viral proteins and other transfection facilitating agents may also beused to advantage. These agents are generally referred to astransfection facilitating agents and as pharmaceutically acceptablecarriers. As used herein, the term gene refers to a segment of nucleicacid which encodes a discrete polypeptide. The term pharmaceutical, andvaccine are used interchangeably to indicate compositions useful forinducing immune responses. The terms construct, and plasmid are usedinterchangeably. The term vector is used to indicate a DNA into whichgenes may be cloned for use according to the method of this invention.

[0080] In another embodiment of this invention, the DNA vaccine encodeshuman influenza virus nucleoprotein, hemagglutinin, matrix,nonstructural, or polymerase gene product. Specific examples of thisembodiment are provided below wherein the human influenza virus geneencodes the nucleoprotein, basic polymerase1, nonstructural protein1,hemagglutinin, matrix1, basic polymerase2 of human influenza virusisolate A/PR/8/34, the nucleoprotein of human influenza virus isolateA/Beijing/353/89, the hemagglutinin gene of human influenza virusisolate A/Texas/36/91, or the hemagglutinin gene of human influenzavirus isolate B/Panama/46/90.

[0081] Data is presented in this specification which exemplifies severaladditional DNA vaccine formulations. More specifically, the presentinvention relates to DNA vaccine formulations which comprise ademetalated solution containing a physiologically acceptable bufferwithin a pH range from at least greater than about 8.0 to about at least9.5, a salt (including but not limited to NaCl, KCl or LiCl) in therange of up to about at 300 mM, and the metal ion chelator EDTA (in therange of up to about 5 mM) in combination with the free radicalscavenger ethanol (in the range of up to about 3%) and the highestappropriate DNA concentration in a sterile glass vial, packaged toprotect the highly purified, nuclease free DNA from light and in aphysiologically acceptable buffer.

[0082] In a specific aspect of the present invention, the DNA vaccineformulations comprise a combination of EDTA and ethanol, NaCl at aconcentration from about 100 mM to about 200 mM, EDTA in the range fromabout 1 μM to about 1 mM, ethanol present up to about 2%, all in thehighest appropriate DNA concentration in a sterile glass vial, packagedto protect the highly purified, nuclease free DNA from light and in aphysiologically acceptable buffer.

[0083] In another embodiment of the DNA vaccine formulations comprisinga combination of EDTA and ethanol, NaCl is present from about 100 mM toabout 200 mM, EDTA is present from about 1 μM to about 750 μM, ethanolis present from about 0.5% to about 2.5%, all in the highest appropriateDNA concentration in a sterile glass vial, packaged to protect thehighly purified, nuclease free DNA from light and in a physiologicallyacceptable buffer which preferably is Tris-HCl at a pH from about 8.0 toabout 9.0. It will be understood that other known buffers with abuffering capacity within various pH ranges, such as pH 8.0 to 9.5, maybe utilized in the various DNA vaccine formulations of the presentinvention. For example, while Tris-HCl is effective up to about pH 9.0,a glycine buffer will in turn be effective at least in the range fromabout pH 9.0 to about 9.5.

[0084] In an especially preferred aspect of the present inventionwherein the DNA vaccine formulations comprise a combination of EDTA andethanol, NaCl at a concentration from about 100 mM to about 200 mM, EDTAis present at about 500 μM, ethanol is present at about 1.0%, all in thehighest appropriate DNA concentration in a sterile glass vial, packagedto protect the highly purified, nuclease free DNA from light and in aphysiologically acceptable buffer which preferably is Tris-HCl at a pHfrom about 8.5 to about 9.0.

[0085] Data is presented which shows that the pH of the formulationaffects the stability of the DNA, and that the optimum pH is ≧8.5. FIG.14 indicates that the highest tested formulation (pH 9.0) provided thegreatest DNA stability was also at the highest pH used (pH 9). Theeffect of buffer type on DNA stability was also performed andcorresponding data is disclosed herein. Briefly, the greatest buffereffect on DNA stability was seen when comparing glycine andBis-Tris-Propane. The glycine buffer at pH 9 was significantly superiorto the Bis-Tris-Propane formulation at the same pH. In contrast, theTris, Bicine and Tricine buffers at pH 8.2 provided nearly identical DNAstability out to 12 weeks. The data correlating buffering to DNAstability suggests controlling free radical oxidation of DNA results inoverall DNA stability then being controlled by formulation pH.

[0086] The effect of light on DNA stability is disclosed herein and itis shown that the detrimental effect of light may be for the most partovercome by the addition of EDTA and ethanol to the formulation. Theaddition of these formulation components stabilizes DNA in samplesstored either in the light or dark. Therefore, DNA vaccine formulationscontaining EDTA and ethanol will be much less sensitive to thedetrimental effects of light and trace metal ions than formulationslacking either of these two stabilizers.

[0087] Various data is also disclosed within this specification whichtests the effect of demetalating buffers prior to generating DNA vaccineformulations. It is disclosed herein demetalation improves DNA stabilityslightly in the formulation containing glycerol, but has no effect onDNA stability in an ethanol formulation. This data shows that the samelevel of DNA stability can be achieved either by controlling freeradical oxidation with succinate and ethanol, or by removal of the tracemetal ions with demetalation. Data is also presented showing that theenhancement of DNA stability by demetalation is effective over a widerange of temperatures and time in storage.

[0088] DNA stability experiments were also performed to show thatethanol is an effective free radical scavenger in the presence of EDTA.The combination of ethanol and EDTA provides a large increase in DNAstability (at pH 7.2) up to 4 weeks, but only a small increase instability by week 8. These results suggest that ethanol is a moreeffective scavenger of free radicals in the presence of EDTA than in itsabsence. Moreover, the results suggest that EDTA alone decreases DNAstability in the absence of ethanol, but increases DNA stability in thepresence of ethanol. These results strongly suggest that ethanol is amore effective scavenger in the presence of EDTA because EDTA removesmetal ions bound to DNA, thereby allowing the generation of hydroxylradicals in the bulk solution, as opposed to the generation of radicalsby iron bound to the DNA. The data presented to exemplify the presentinvention also shows that the DNA stabilizing effects of ethanol andEDTA/EtOH are greater at pH 8.0 than at pH 7.2. It is shown further thata similar degree of protection can be obtained from the combination ofsuccinate and ethanol.

[0089] Another exemplified portion of the invention relates toalternative metal ion chelators, including but not necessarily limitedto NTA (nitrilotriacetic acid) and DTPA (diethylenetriaminepentaaceticacid). Data is presented which shows that NTA or DTPA, preferably DTPA,enhance DNA stability in the absence of ethanol.

[0090] The present invention relates to either liquid or lyophilized DNAvaccine formulations. Data is presented which shows that stability ofthe best lyophilized formulation exceeded that of the liquidformulations over the short term whereas liquid formulations showed goodstability over longer periods of time. The results also show thatdemetalation improves the stability of the lyophilized DNA informulations 1 and 2, but has little effect on the % SC DNA in the otherlyophilized formulations. Therefore, lyophilization is an effectivemethod for stabilizing DNA vaccines and demetalation of the formulationbuffer improves the stability of lyophilized DNA in some formulations.

[0091] The following examples are provided to further define theinvention, without limiting the invention to the specifics of theexamples.

EXAMPLE 1

[0092] V1J Expression Vector—V1J is derived from vectors V1 and pUC18, acommercially available plasmid. V1 was digested with SspI and EcoRIrestriction enzymes producing two fragments of DNA. The smaller of thesefragments, containing the CMVintA promoter and Bovine Growth Hormone(BGH) transcription termination elements which control the expression ofheterologous genes, was purified from an agarose electrophoresis gel.The ends of this DNA fragment were then “blunted” using the T4 DNApolymerase enzyme in order to facilitate its ligation to another“blunt-ended” DNA fragment.

[0093] pUC18 was chosen to provide the “backbone” of the expressionvector. It is known to produce high yields of plasmid, iswell-characterized by sequence and function, and is of minimum size. Weremoved the entire lac operon from this vector, which was unnecessaryfor our purposes and may be detrimental to plasmid yields andheterologous gene expression, by partial digestion with the HaeIIrestriction enzyme. The remaining plasmid was purified from an agaroseelectrophoresis gel, blunt-ended with the T4 DNA polymerase, treatedwith calf intestinal alkaline phosphatase, and ligated to theCMVintA/BGH element described above. Plasmids exhibiting either of twopossible orientations of the promoter elements within the pUC backbonewere obtained. One of these plasmids gave much higher yields of DNA inE. coli and was designated V1J. This vector's structure was verified bysequence analysis of the junction regions and was subsequentlydemonstrated to give comparable or higher expression of heterologousgenes compared with V1.

EXAMPLE 2

[0094] Influenza Virus Gene Constructs In Expression Vector V1j- Many ofthe genes from the A/PR/8/34 strain of influenza virus were cloned intothe expression vector V1J, which gives rise to expression at levels ashigh or higher than in the V1 vector. The PR8 gene sequences are knownand available in the GENBANK database. For each of the genes clonedbelow, the size of the fragment cloned was checked by sizing gel, andthe GENBANK accession number against which partial sequence was comparedare provided. For a method of obtaining these genes from virus strains,for example from virus obtained from the ATCC (A/PR/8/34 is ATCC VR-95;many other strains are also on deposit with the ATCC).

[0095] A. Subcloning the PR8 Genes into V1J:

[0096] 1. NP—The NP gene was subcloned from pAPR501 (J. F. Young, U.Desselberber, P. Graves, P. Palese, A. Shatzman, and M. Rosenberg(1983), in The Origins of Pandemic Influenza Viruses, ed. W. G. Laver,(Elsevier, Amsterdam) pp.129-138). It was excised by cutting pAPR501with EcoRI, the fragment gel purified, and blunted with T4 DNAPolymerase. The blunted fragment was inserted into V1J cut with Bgl IIand also blunted with T4 DNA Polymerase. The cloned fragment was 1.6kilobases long.

[0097] 2. NS—The NS gene was subcloned from pAPR801 (J. F. Young, U.Desselberber, P. Graves, P. Palese, A. Shatzman, and M. Rosenberg(1983), in The Origins of Pandemic Influenza Viruses, ed. W. G. Laver,(Elsevier, Amsterdam) pp.129-138). It was excised by cutting pAPR801with EcoRI, the fragment gel purified, and blunted with T4 DNAPolymerase. The blunted fragment was inserted into V1J cut with Bgl IIand also blunted with T4 DNA Polymerase. The cloned fragment was 0.9kilobases long (the complete NS coding region including NS1 and NS2).

[0098] 3. HA—The HA gene was subcloned from pJZ102 (J. F. Young, U.Desselberber, P. Graves, P. Palese, A. Shatzman, and M. Rosenberg(1983), in The Origins of Pandemic Influenza Viruses, ed. W. G. Laver,(Elsevier, Amsterdam) pp.129-138). It was excised by cutting pJZ102 withHind III, the fragment gel purified, and blunted with T4 DNA Polymerase.The blunted fragment was inserted into V1J cut with Bgl II and alsoblunted with T4 DNA Polymerase. The cloned fragment was 1.75 kilobaseslong.

[0099] 4. PB1—The PB1 gene was subcloned from pGem1-PB1 (The 5′ and 3′junctions of the genes with the vector were sequenced to verify theiridentity. See J. F. Young, U. Desselberber, P. Graves, P. Palese, A.Shatzman, and M. Rosenberg (1983), in The Origins of Pandemic InfluenzaViruses, ed. W. G. Laver, (Elsevier, Amsterdam) pp.129-138). It wasexcised by cutting pGem-PB1 with Hind III, the fragment gel purified,and blunted with T4 DNA Polymerase. The blunted fragment was insertedinto V1J cut with Bgl II and also blunted with T4 DNA Polymerase. Thecloned fragment was 2.3 kilobases long.

[0100] 5. PB2—The PB2 gene was subcloned from pGem1-PB2 (The 5′ and 3′junctions of the genes with the vector were sequenced to verify theiridentity. See J. F. Young, U. Desselberber, P. Graves, P. Palese, A.Shatzman, and M. Rosenberg (1983), in The Origins of Pandemic InfluenzaViruses, ed. W. G. Laver, (Elsevier, Amsterdam) pp.129-138). It wasexcised by cutting pGem-PB2 with BamH I, and gel purifying the fragment.The sticky-ended fragment was inserted into V1J cut with Bgl II. Thecloned fragment was 2.3 kilobases long.

[0101] 6. M1—The M1 gene was generated by PCR from the plasmid p8901MITE. The M sequence in this plasmid was generated by PCR from pAPR701(J. F. Young, U. Desselberber, P. Graves, P. Palese, A. Shatzman, and M.Rosenberg (1983), in The Origins of Pandemic Influenza Viruses, ed. W.G. Laver, (Elsevier, Amsterdam) pp.129-138.). The PCR fragment was gelpurified, cut with Bgl II and ligated into V1J cut with Bgl II. Thecloned fragment was 0.7 kilobases long. The amino terminus of theencoded M1 is encoded in the “sense” primer shown above as the “ATG”codon, while the M1 translation stop codon is encoded by the reverse ofthe “TCA” codon, which in the sense direction is the stop codon “TGA”.

[0102] B. Influenza Gene-V1J Expression Constructs

[0103] In each case, the junction sequences from the 5′ promoter region(CMVintA) into the cloned gene is shown. The position at which thejunction occurs is demarcated by a “/”, which does not represent anydiscontinuity in the sequence. The method for preparing these constructsis summarized after all of the sequences below. Each sequence providedrepresents a complete, available, expressible DNA construct for thedesignated influenza gene.

[0104] Each construct was transiently transfected into RD cells, (ATCCCCL136), a human rhabdomyosarcoma cell line in culture. Forty eighthours after transfection, the cells were harvested, lysed, and westernblots were run (except for the V1J-PR-HA construct which was tested inmice and gave anti-HA specific antibody before a western blot was run,thus obviating the need to run a western blot as expression was observedin vivo). Antibody specific for the PB1, PB2 and NS proteins wasprovided by Stephen Inglis of the University of Cambridge, who usedpurified proteins expressed as β-galactosidase fusion proteins togenerate polyclonal antisera. Anti-NP polyclonal antiserum was generatedby immunization of rabbits with whole A/PR/8/34 virus. Anti-M1 antibodyis commercially available from Biodesign as a goat, anti-fluA antiserum,catalog number B65245G. In each case, a protein of the predicted sizewas observed, confirming expression in vitro of the encoded influenzaprotein.

[0105] The nomenclature for these constructs follows the convention:“Vector name-flu strain-gene”. In every case, the sequence was checkedagainst known sequences from GENBANK for the cloned and sequencedA/PR/8/34 gene sequence.

[0106] C. Production of V1jns

[0107] An Sfi I site was added to V1Jneo to facilitate integrationstudies. A commercially available 13 base pair Sfi I linker (New EnglandBioLabs) was added at the Kpn I site within the BGH sequence of thevector. V1Jneo was linearized with Kpn I, gel purified, blunted by T4DNA polymerase, and ligated to the blunt Sfi I linker. Clonal isolateswere chosen by restriction mapping and verified by sequencing throughthe linker. The new vector was designated V1Jns. Expression ofheterologous genes in V1Jns (with Sfi I) was comparable to expression ofthe same genes in V1Jneo (with Kpn I).

EXAMPLE 3

[0108] Growth of Microbial Cells, Cell Lysis and Clarification—One literof frozen E. coli cell slurry was used to make 8 liters of cellsuspension in STET buffer (8% sucrose, 0.5% TRITON, 50 mM TRIS buffer,pH 8.5 and 50 mM EDTA). The absorbance of the cell suspension at 600 nmwas about O.D. 30. The suspension was stirred continuously to ensurehomogeneity. The viscosity of the cell suspension was measured to beabout 1.94 cp at room temperature (24° C.). The cell suspension waspumped through the heat exchanger at 81 mL/min which corresponded to aresidence time of the cell solution in the heat exchanger of about 35seconds. The bath temperature was maintained at 92° C. The inlet andoutlet temperatures of the cell solution were measured to be about 24°C. and about 89° C. (average), respectively. About 1 liter of sample wasrun through the heat exchanger. No visible clogging of the tube wasobserved although the lysate was somewhat thicker than the startingmaterial. The lysate was cooled to room temperature and its viscositywas measured to be about 40 cp. The cell lysate was clarified by batchcentrifugation at 9000 RPM for 50 minutes using the Beckman J-21.Analysis of the supernatant confirmed effective cell lysis and productrecovery. The product yield produced by flow-through heat lysis was atleast comparable to that made by the Quigley & Holmes boiling method.The latter method; however, must be carried out at the laboratory scalein batch mode and is therefore unsuitable for large-scale (5 liters orgreater) processing. Since the heat exchanger process is flow-through,there is no maximum limit to the volume of cell suspension that can beprocessed. This process can therefore accomodate very large scalefermentations of bacteria to produce large quantities of highly purifiedplasmid DNA.

[0109] The clarified lysate was then filtered through a membrane havinga pore size of 0.45 microns to remove finer debris. The filtrate wasthen diafiltered using a membrane having a molecular weight cutoff ofabout 100,000.

EXAMPLE 4

[0110] Control and Reproducibility of Cell Lysis with the HeatExchanger—Adjusting the flowrate (i.e., residence time) at which thecell slurry is pumped through the heat exchanger permits tight controlof the temperature of lysis, i.e., the outlet temperature. A cell slurrysolution was prepared as described in Example 3 and pumped through theheat exchanger at flow rates ranging from 160 to 850 mL/min. Thecorresponding outlet temperatures ranged between 93° C. and 65° C.,respectively. The initial temperature of the cell slurry was 24° C. andthe bath temperature was kept constant at 96° C. In addition, a numberof runs were performed where an outlet temperature of 80° C. wastargeted. Yields of 24 mg of circular DNA per L of clarified supernatantwere consistently obtained demonstrating the reproducibility of theprocess.

EXAMPLE 5

[0111] Purification of Plasmid DNA—Microbial cells and lysates wereprepared as described, and the following analyses were performed. Toillustrate that the addition of 100 mM EDTA vs 50 mM EDTA increased thepercentage of supercoiled DNA, and to determine an acceptable range ofoutlet temperatures (i.e., lysis temperature) with respect to recoveryof supercoiled DNA, the following analyses were performed. Thesupercoiled form of plasmid DNA is desirable since it is more stablethan the relaxed circle form. One way that supercoiled DNA can beconverted to open circle is by nicking with DNase. We found that theaddition of 100 mM EDTA vs 50 mM in the STET buffer minimized theformation of open circle plasmid. The cell suspension was prepared asdescribed. The operating flow rate for these runs was approximately 186ml/min. The temperatures of the inlet, outlet and bath are 24° C., 92°C. and 96° C. respectively.

[0112] An acceptable range of lysis temperatures was determined bymeasuring the percentage of supercoiled plasmid generated for each run.The concentration of supercoiled plasmid as a function of exittemperature. An acceptable range of lysis temperatures is between 75° C.and 92° C. At temperatures below 75° C., more relaxed circle plasmid wasgenerated, most likely due to increased DNase activity. Above 93° C.,the levels of supercoiled plasmid appear to diminish, possibly due toheat denaturation.

[0113] Following continuous heat lysis and centrifugation, 1 mL ofclarified lysate was either incubated with 5 μg RNase for 2 hours, orwas used untreated. The RNase treated and untreated samples were thenloaded onto an anion exchange column (Poros Q/M 4.6×100) that had beenpreviously equilibrated with a 50-50 mixture of solvents A and B [HPLCsolvent A: 20 mM Tris/Bis Propane, pH 8.0; and solvent B: 1 M NaCl in 20mM Tris/Bis Propane, pH 8.0]. The column was eluted using a gradient of50% to 85% B run over 100 column volumes. Open circle plasmid elutes atapproximately 68% B and supercoiled elutes at 72% B.

[0114] As described above, diafiltration prior to anion exchangechromatography greatly increases the amount of lysate that can be loadedonto the column.

[0115] The plasmid DNA eluted from the anion exchange column wasseparated into the individual forms by reversed phase HPLC analysis. Theforms are supercoiled plasmid (form 1) and nicked circle (form 2). Thetwo forms were easily separated and allowed the isolation of individualforms of the plasmid.

EXAMPLE 6

[0116] Highly Purified Plasmid DNA From a Chromatography-based Process—Afermentation cell paste was resuspended in modified STET buffer and thenthermally lysed in a batchwise manner. Alternatively a fermentation cellpaste is resuspended in modified STET buffer and then thermally lysed inthe flow-through process described above. The lysate was centrifuged asdescribed above. Twenty ml of the supernatant were filtered as describedabove and loaded onto an anion exchange column (Poros Q/M 4.6×100) thatwas previously equilibrated with a 50-50 mixture of buffers A and Bdescribed above. A gradient of 50% to 85% B was run over 50 columnvolumes with a flow rate of 10 ml/minute. Fractions of 2.5 ml each werecollected from the column. The supercoiled plasmid DNA eluted from thecolumn at 72% B.

[0117] The anion exchange product was then loaded onto a reversed phasechromatography column (Poros R/H) which had been previously equilibratedwith 100 mM ammonium bicarbonate at pH 8.0, and a gradient of 0% to 80%methanol was used to elute the bound material. The highly purifiedsupercoiled plasmid DNA eluted at 22% methanol.

[0118] Based on the agarose gels and the colorimetric and HPLC assaysdescribed, the final product, shown in FIG. 9, is highly pure. Theproduct consists of greater than 90% supercioled and less than 10% opencircle plasmid. RNA was below the limits of detection of the assay used.Genomic DNA and protein contaminant levels were also below the limits ofdetection in the assays used. The overall supercoiled plasmid yield atthe end of the process was approximately 60% of the supercoiled plasmidin the clarified lysate.

EXAMPLE 7

[0119] Multi-Gram Scale Purification of Plasmid DNA—4.5 L of frozen E.coli cell slurry was used to make 33.7 L of cell suspension in STETbuffer (8% sucrose, 2% Triton, 50 mM Tris buffer, 50 mM EDTA, pH 8.5)with 2500 units/ml of lysozyme. The absorbance of the suspension at 600nm was about O.D. 30. The suspension was stirred at room temperature for15 minutes to ensure proper mixing and then was incubated for 45 minuteswith continuous stirring at 37° C. Following incubation, mixing wascontinued at room temperature and the cell suspension was pumped throughthe heat exchanger at a flowrate of 500 ml/min. The batch temperaturewas maintained at 100° C. and the inlet and outlet temperatures of thecell suspension were measured to be about 24° C. and between 70-77° C.,respectively. The cell lysate exiting the heat exchanger was collectedin Beckman centrifuge bottles (500 mls each) and the material wascentrifuged immediately in Beckman J-21 centrifuges for 50 minutes at9000 RPM. Following centrifugation, the supernatant was found to contain4-5 times more plasmid product than in the case without lysozymeincubation. The supernatant product of the centrifugation wasimmediately diafiltered against 3 volumes of TE buffer (25 mM Tris-EDTAat pH 8.0) and then incubated with 20×10⁵ units of E. coli RNase for 2-4hours at room temperature. After completion of the incubation, theproduct solution was then diafiltered an additional 6 volumes with TEbuffer using a 100 kD MWCO membrane and then filtered through a 0.45micron filter to remove residual debris. The filtered lysate was dilutedto 0.7 M NaCl with a 20 mM Bis/Tris Propane-NaCl buffer at pH 7.5, whichprepares the diluted filtrate for loading onto the anion exchangecolumn. The anion exchange column (3.6 L of POROS PI/M) was previouslyequilibrated with 20 mM Bis/Tris Propane and 0.7M NaCl. The filteredlysate was loaded to column capacity. In this case 5 grams ofsupercoiled plasmid was loaded onto the anion exchange column. Afterloading, the column was washed with 2-4 column volumes of 20 mM Bis/TrisPropane and 0.7 M NaCl. A 10 column volume gradient from 0.7 M NaCl to2.0 M NaCl in 20 mM Bis/Tris Propane was performed to clear most of theE. coli protein. RNA and some endotoxin. The supercoiled plasmidfraction eluted between 1.4 M and 2.0 M NaCl. The supercoiled fractionfrom the anion exchange column, which contained 4 grams of supercoiledplasmid was then diluted 2-3 times with pyrogen free water, adjusted to1.2% IPA and pH adjusted to 8.5 with 1 N NaOH. The diluted anionexchange supercoiled fraction was then loaded onto a 7 L reversed phasecolumn (POROS R2/M) which had been previously equilibrated with 100 mMAmmonium Bicarbonate containing 1.2% IPA. In this case, 3.2 grams ofsupercoiled plasmid were loaded onto the reversed phase column and thenthe column was washed with 6-10 column volumes of 1.2% IPA in 100 mMAmmonium Bicarbonate. This extensive wash was performed to clearimpurities. Next, a gradient of 1.2% IPA to 11.2% IPA in 5 columnvolumes was performed. The supercoiled plasmid fraction elutes at about4% EPA. The supercoiled product fraction from the reversed phase columnwas then concentrated and diafiltered into normal saline using a 30 kDMWCO membrane. The final product bulk was filtered through a 0.22 micronfilter. The overall product yield of the process was more than 50% ofthe supercoiled plasmid in the clarified cell lysate as indicated by theanion exchange HPLC assay.

EXAMPLE 8

[0120] Polynucleotide Vaccination in Primates

[0121] Antibody to NP in Rhesus monkeys—Rhesus monkeys (006 NP, 009 NPor control 101; 021) were injected with 1 mg/site of RSV-NP i.m. in 3sites on day 1. Injections of 1 mg each of RSV-LUX and CMV-int-LUX,constructs for the reporter gene firefly luciferase expression, weregiven at the same time into separate sites. Animals were re-injected onday 15 with the same amounts of DNA as before and also with 1 mg ofpD5-CAT, a construct for the reporter gene chloramphenical acetyltransferase expression, in 1 site each. Muscle sites containing reportergenes were biopsied and assayed for reporter gene activity. Serum wascollected 3, 5, 9, 11, 13, and 15 weeks after the first injection. Thefirst positive sample for anti-NP antibody was collected at week 11 andpositive samples were also collected on weeks 13 and 15. Anti-NPantibody was determined by ELISA.

[0122] Hemagglutination inhibiting (HI) antibody in rhesusmonkeys—Monkeys were injected i.m. with V1J-PR-HA on day 1. Two animalseach received 1 mg, 100 μg, or 10 μg DNA in each quadriceps muscle. Eachinjection was administered in a volume of 0.5 ml. Animals were bledprior to injection on day 1. All animals were reinjected with DNA on day15, and blood was collected at 2-4 week intervals thereafter.Hemagglutination inhibition (HI) titers against A/PR/8/34 were positiveat 5 weeks, 9 weeks and 12 weeks after the first injection of V1J-PR-HADNA.

EXAMPLE 9

[0123] Effect of container type and DNA concentration on DNAstability—The conversion of supercoiled plasmid DNA to its open circularand linear forms is a major physicochemical change occurring during invitro storage. In Examples 9-16 the effects of a variety of storageconditions on the stability of plasmid DNA were determined by monitoringthe chemical cleavage of the phosphodiester backbone, leading to theconformational conversion of supercoiled plasmid DNA to open circularand linear forms. To monitor this change, plasmid DNA (Influenza DNAvaccine, HA (Georgia/93) was formulated, sterilized by sterilefiltration if necessary, placed in sterile capped glass vials (0.8 mL in3 mL vials) and incubated at various temperatures. Following theincubation period, a single vial of each particular formulation wasremoved from the incubator and frozen at minus 70° C. Each vial was thenthawed and 20 ng of DNA from the vial was applied to a 1% agarose geland electrophoresed for 90 minutes. The gel was then stained withethidium bromide, destained and photographed under UV illumination. Toquantitate the amount of supercoiled, open circle and linear DNA in eachsample, the negative of the gel photograph was scanned with a Bio-RAD(GS-670) densitometer. The absorbance data for each lane on the gel wasthen compared to the absorbance of a series of supercoiled, open circleand linear DNA standards on the same gel using a computer program(Molecular Analyst, version 1.3, BIO-RAD Laboratories). The absorbanceof the DNA standards was used to construct a standard curve forsupercoiled, open circle and linear DNA. Seven DNA standards of eachform were applied to the gel (1 to 30 ng of supercoiled DNA, 0.1875 to15 ng of open circle and linear DNA). To express the stability of theDNA over time, the amount of supercoiled DNA in the sample at time zerowas normalized to 100% (the initial % supercoiled DNA normally rangedfrom 90-100%). The stability of the DNA samples was then expressed asthe percent of initial supercoiled DNA remaining after some period ofincubation. Although the were each determined from sampling a singlevial, multiple time points were usually taken to allow the observationof a decreasing percent supercoiled DNA remaining over an extendedperiod of time. For samples incubated at 50° C. the sampling time pointswere usually at 1, 2, 4, 6 and 8 weeks, while the 4, 25 and 37° C.samples were analyzed after 1,2,3 and sometimes 6 months.

[0124] Using chromatographically purified plasmid DNA, preformulationexperiments were initiated to determine the potential mechanism ofplasmid degradation during in vitro storage under a variety of storageconditions. To examine the stability of the Influenza DNA vaccine(Georgia/93) during exposure to different container types plasmid DNA(100 mcg/mL in saline) was incubated in different containers for 6months at 5, 24 and 37° C. The results (FIGS. 1A, 1B, 1C and 1D)indicated that glass vials were superior to siliconized glass,autoclaved plastic or gamma radiated plastic vials for stabilizing theDNA. To determine the effect of DNA concentration on stability, plasmidDNA at 20, 100 and 1800 mcg/mL in saline was incubated for 6 months at37° C. An analysis of the supercoiled DNA content indicated thatstability was dose dependent, with higher concentrations of plasmidbeing more stable during storage at 37° C. (FIG. 2). Further studies onthe effect of DNA concentration, with the DNA formulated in saline orPBS, also indicated that higher concentrations were more stable at 2 to8° C. (Table 1A) and at minus 70° C. (Table 1B). TABLE 1 Stability datafor Influenza DNA vaccine, HA (Georgia/93) formulations stored at (A) 2to 8° C. and (B) minus 70° C. (A) Formulation/Lot # Buffer¹ Test initial1 month 3 month High mcg/mL² Saline % SC 94 94 93 V510-HSS-001-C001 pH6.6 6.6 6.5 V510-PBS-1.0 PBS % SC 96 95 96 pH 7.3 7.3 7.3 20 mcg/mLSaline % SC 91 66 63 V510-HSS-001-B002 pH 6.7 6.3 6.3 V510-HSS-003-A002PBS % SC 91 93 90 pH 7.2 7.2 7.2 2 mcg/mL Saline % SC 87 67 48V510-HSS-001-B001 pH 6.3 6.0 6.5 V510-HSS-003-B002 PBS % SC 93 92 91 pH7.2 7.1 7.2 ¹0.9% Saline, adj. to pH 7.2; PBS: 6 mM Phosphate BufferContaining 150 mM Saline, pH 7.2 ²High mcg/mL: Saline formulated at2,200 mcg DNA/mL, PBS formulated at 1,000 mcg DNA/mL % SC = %supercoiled (B) Formulation/Lot # Buffer¹ Test initial 1 3 High mcg/mL²Saline % SC 94 NS 91 V510-HSS-001-C001 pH 6.6 NS 6.4 V510-PBS-1.0 PBS %SC 96 94 97 pH 7.3 7.3 7.4 20 mcg/mL Saline % SC 91 86 84V510-HSS-001-B002 pH 6.7 NS 6.3 V510-HSS-003-A002 PBS % SC 91 95 95 pH7.3 7.2 7.2 2 mcg/mL Saline % SC 87 83 76 V510-HSS-001-B001 pH 6.3 6.16.6 V510-HSS-003-B002 PBS % SC 93 93 95 pH 7.2 7.2 7.2 ¹0.9% Saline,adj. to pH 7.2; PBS: 6 mM Phosphate Buffer Containing 150 mM Saline, pH7.2 ²High mcg/mL: Saline formulated at 2,200 mcg DNA/mL, PBS formulatedat 1,000 mcg DNA/mL NS: Not scheduled

[0125] DNA stability experiments show that the rubber stoppers used tocap the glass vial containers have a detrimental effect on DNAstability. Table 2 shows the effects of three stopper types on DNAstability in two different DNA vaccine formulations. The vials(containing the DNA solutions, 2 mcg/mL) were incubated at 50° C. inboth the right-side up and up-side down configuration to determine theeffect of the stoppers on DNA stability. The DNA was more stable whenthe vials were incubated in the right-side up configuration, for eachstopper type tested. Furthermore, the addition of ethanol enhances DNAstability and reduces the degradative effect of the stopper. For stopper#3 (Table 2), the addition of ethanol totally eliminated the degradativeeffect of the stopper on DNA stability. These results suggest that thestopper contributes to DNA degradation during storage, and that ethanolcan be used to control the degradative effects of some stopper types, onDNA stability.

[0126] To determine how much the stopper influences the stability of DNAin glass vials an experiment was carried out to determine the stabilityof DNA over 8 weeks at 50° C., in glass vials with three different typesof stoppers and in sealed glass ampules. Table 3 shows formulations withand without succinate (a DNA stability enhancer), the DNA wassignificantly more stable in sealed glass ampules than in glass vialswith any of the tested stoppers. Also, there were only small differencesin DNA stability among the vials with the three different stopper types.These data show that different stopper types in four differentformulations (Tables 2, 3) contribute significantly to DNA degradation.The optimization of DNA stability in glass vials will require thetesting of several stopper types, and may also require the addition offree radical scavengers like ethanol to control to degradative effectsof the stopper. TABLE 2 Summary of the effects of stopper type on DNAstability, in formulations with and without ethanol. Percent of initialsupercoiled DNA was determined by agarose gel electrophoresis. % ofInitial supercoiled DNA remaining 25-Apr 9-May 23-May 20-Jun ConditionsInitial % SC 2 weeks 4 weeks 8 weeks C1-up* 92 85 40 21 C1- 92 72 18 0down** C2-up 95 81 46 26 C2-down 95 77 3 0 C3-up 93 26 8 0 C3-down 93 3010 0 C4-up 95 93 64 47 C4-down 95 86 39 25 C5-up 95 92 71 45 C5-down 9586 62 34 C6-up 95 94 78 71 C6-down 95 93 71 71

[0127] TABLE 3 Stability of DNA in glass ampules and vials with threestopper types, using formulations with and without succinate. Percent ofinitial supercoiled DNA was determined by agarose gel electrophoresis. %of Initial Supercoiled DNA Remaining 8-Oct 22-Oct 5-Nov 19-Nov 3-DecConditions Initial % SC 2 weeks 4 weeks 6 weeks 8 weeks C1 93 38 19 7 7C2 93 53 18 7 3 C3 93 53 23 5 2 C4 93 61 42 32 19 C5 94 59 37 27 11 C694 60 37 17 3 C7 94 66 37 16 5 C8 94 68 60 48 39

EXAMPLE 10

[0128] Effect of salt concentration on DNA stability—To examine theeffect of salt concentration on DNA stability an initial experiment wasset up to determine the melting temperature (Tm) of the DNA over a rangeof NaCl concentrations from 0 to 200 mM. To determine the Tm at eachconcentration of NaCl, the UV absorbance of plasmid DNA (10 mcg/mL DNA)formulated in 10 mM sodium phosphate (pH 6.0) was monitored at 260 nm asthe temperature was increased. The results (FIG. 3) indicated a Tm of72°, 73°, 78° and 82° C. for 0, 5, 20 and 50 mM NaCl, respectively. TheTm value for 100 and 200 mM NaCl could not be determined because thestrong stabilizing effect of the salt elevated the Tm to greater than90° C. These results suggest that the minimum NaCl concentrationnecessary to provide the maximum DNA thermal stability is in the 100-200mM range.

[0129] To examine the effect of NaCl concentration on the rate ofconversion of supercoiled to open circle DNA during storage, plasmid DNA(100 mcg/mL) was formulated in 10 mM sodium phosphate buffer (pH 7.2)with NaCl varying from 1 to 320 mM. The solutions were incubated at 60°C. for 48 hours and analyzed by agarose gel electrophoresis. The results(FIG. 4) indicated an increase in DNA stability as NaCl concentrationwas increased from 1 to 160 mM and no significant difference instability between 160 and 320 mM. These results are consistent withthose of FIG. 3, suggesting that the minimum NaCl concentrationnecessary for maximum DNA stability is between 100-200 mM.

[0130] To examine the effect of divalent cations on DNA stability,plasmid DNA was formulated in either TE, phosphate buffered saline (PBS)or saline (0.9% NaCl) in the presence of ZnCl₂, CaCl₂, MnCl₂ or MgCl₂.The DNA solutions (100 mcg/mL) were incubated at 60° C. for 48 hours andanalyzed by agarose gel electrophoresis. The results (FIGS. 5A, 5B and5C) indicated that zinc ions did not improve DNA stability. However, theeffect of calcium ions was variable. In TE and saline, calcium ionsimproved stability, while in PBS calcium ions did not increasestability. Manganese ions improved the stability in TE, but only at lowconcentrations. In PBS and saline, manganese ions did not improvestability. Magnesium ions increased DNA stability in all threeformulations, but only at high concentrations in saline.

EXAMPLE 11

[0131] Effect of buffer type and pH on DNA stability—Historically,plasmid DNA has been stored in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0)with routine molecular biology manipulations being carried out in salineand distilled water. Consequently, we deemed it necessary to examine anddefine an appropriate buffer in which to both store plasmid DNA andmanipulate it for future formulation experiments. As an initial study,DNA was incorporated into different buffers at various pH values, andstored at different temperatures. Plasmid DNA was formulated at 500mcg/mL in PBS at pH 4.5, 7.2 and 9.0, TE at pH 8.0 and 10, distilledwater (pH 6.0). saline (pH 6.0) and TFA (trifluoroacetic acid; 0.1%) atpH 2.0. Samples were stored at 4° and 24° C. then analyzed at 1, 4 and12 week intervals by agarose gel electrophoresis. TABLE 4 1 Week 4 Week12 Week Buffer 4C. 24C. 4C. 24C. 4C. 24C. PBS, pH 9.0 + + + + + + PBS,pH 7.2 + + + + + + PBS, pH 4.5 + + + + + − TE, pH 10 + + + + + + TE, pH8.0 + + + + + + deionized H₂O + + + +/− +/− − TFA (0.1%) +/− − − − − −saline + + + + + −

[0132] Table 4 shows the conformational stability of DNA plasmid storedat various pH's. Samples were analyzed by agarose gel electrophoresis;(+) supercoiled, (+/−) supercoiled/open circle, (−) mainly open circle(nicked plasmid). These data show that at low pH (2.0-4.5) DNA israpidly degraded while at high pH (7.2-9.0) there is little degradation.For example, plasmid DNA stored in both TE and PBS at pH values from 7to 10, was stable up to 12 weeks at 4° C. To further investigate this pHeffect on the conformational stability of DNA plasmid, a UV meltingcurve was generated over the pH range of 4.5-10 (FIG. 6). This techniqueallows one to determine the relative stability of DNA at various pHs bymeasuring the melting point transition (Tm), which occurs as a result ofbase pair unpairing leading to increased absorbance at 260 nm. Theresults show that as the pH decreases the absorbance of the DNA, at 260nm, increases at a lower temperature, thus a lower Tm. This indicatesthat double stranded DNA is less ordered at lower pHs, and that theunpaired regions of the molecule are more susceptible to breakage atlower pH. One other point to emphasize is that the plasmid DNA was quiteresistant to heat denaturation under these conditions. Since heating to90° C. did not cause complete denaturation of double stranded DNA, aprecise Tm could not be determined.

[0133] To quickly explore storage conditions best suited for long termDNA storage, accelerated stability studies were established in whichsamples were stored at 60° C. for 48 hours and then analyzed by agarosegel electrophoresis. Previous results (Table 1) indicate that storagebelow pH 6.0 is problematic to DNA stability and either salt or EDTA (orboth) may be necessary to stabilize it toward conformationaldegradation. Thus two buffers, PBS and TE (Tris-EDTA), were analyzed fortheir ability to stabilize plasmid in the pH range of 6.0-8.5. PlasmidDNA samples were formulated at 100 mcg/mL in the appropriate buffer.Starting material contained 90% of the supercoiled species. The resultsare shown in FIG. 7A. After incubation at 60° C. for 48 hours, samplesstored in TE at pH 7.5-8.5 had 10-25% supercoiled plasmid remainingwhile those formulated in PBS (pH 7.5-8.5) contained 50% supercoiledplasmid. Furthermore, TE had no apparent stabilizing effect on the DNAat pH 6.5-7.0, while PBS did show a slight stabilizing effect in thesame pH range. Neither buffer prevented degradation at pH 6.0.

[0134] The results from accelerated studies have indicated that both pHand salt are important parameters to optimize, in order to stabilize DNAduring storage. These preliminary results suggested that PBS is a betterstabilizer than TE. However, it was not clear whether the stabilizingeffect of PBS was due to the presence of phosphate ions, or due to thepresence of NaCl. Furthermore, the question of whether a metal ionchelator would stabilize DNA needed to be more adequately addressed. Toanswer these questions an accelerated stability study (60° C., 48 hrs,100 mcg/mL DNA) was implemented in which PBS was supplemented with EDTAor additional phosphate, and TE was supplemented with NaCl or phosphate.The results shown in Table 3 indicate that the addition of EDTA orphosphate to PBS did not enhance the stability of DNA to degradation.However, the addition of 150 mM NaCl to TE buffer increased thestability of DNA when compared to non supplemented TE. These resultssuggest that the stabilizing effect of PBS over TE was due to thepresence of 150 mM NaCl in the PBS. TABLE 5 Formulation % supercoiledDNA remaining PBS (pH 7.2) 50 PBS + 1 mM EDTA 50 PBS + 20 mM sodiumphosphate 50 PBS + 100 mM sodium phosphate 50 PBS (pH 6.0) 0 PBS (pH10.0) 50 TE + 20 mM sodium phosphate 10 TE 10 Saline (pH 8.0) 0 TE + 150mM NaCl 50 PBS + 10 mM EDTA 50

[0135] Table 5 shows the conformational stability of plasmid DNA at 60°C., for 48 hours in various buffers. At time zero, samples contained 90%supercoiled DNA. Since the presence of NaCl in the PBS appeared to bethe reason for improved DNA stability, compared to TE, another study wasinitiated to compare saline to PBS. For these experiments, plasmid DNA(chromatographically pure) was directly formulated at high concentration(2.0-2.5 mg/mL) in 0.9% NaCl. Probe stability studies using this DNAwere performed with a low concentration of plasmid (2 mcg/mL) formulatedin either a PBS buffer (pH 7.2) or 0.9% NaCl adjusted to pH 6, 7, and 8.The plasmid's supercoiled DNA content was monitored by agarose gelelectrophoreses over three months at 25° and 37° C. As shown in FIG. 8A(37° C.) and 8B (37° C.), when the initial pH of saline formulatedmaterial is decreased, the rate of loss of supercoiled plasmid DNAincreased. The TABLE 6 Table 6: Summary of stability probes 3-9; toexamine the effect of buffer ions and pH on the stability of InfluenzaDNA vaccine, HA (Georgia/93) during storage at 37° C. Percent of initialsupercoiled DNA remaining was determined by agarose gel electrophoresis.Percent of initial supercoiled DNA remaining after three months at 37°C. Probe # Formulation* [DNA]mcg/mL 3 months, 37° C. 3 control 20 36 3PBS, pH 7.8 100 63 4 control 20 0 4 control 100 0 4 saline (0.9% (w/v)NaCl) + 20 36 10 mM sodium succinate, pH 7.2 5 Control 20 57 5 saline +10 mM sodium 20 64 succinate, pH 7.2 6 control 20 55 6 saline + 10 mMsodium 20 80 bicarbonate, pH 7.2 7 control 2 29 8 PBS, pH 6.0 20 0 8control 20 37 8 PBS, pH 8.0 20 0 9 control 2 31 9 PBS, pH 8.0 2 36

[0136] saline formulated material did not maintain pH over time. Forexample, after 2 months at 37° C. the saline formulated plasmid (initialpH of 6, 7 and 8) had a pH value of 5.9, 6.0 and 6.3, respectively. Incontrast, the PBS formulated material maintained its pH value (7.1-7.2)over the same incubation period. The PBS formulated material alsocontained the highest percentage of supercoiled content with a more orless consistent level of supercoiled plasmid after 3 months at 25° C.

[0137] As the supercoiled content of plasmid decreased, a concomitantincrease in the open circle form of the plasmid was observed by agarosegel electrophoresis. After longer periods of time, the open circularform converts to linear plasmid DNA. For example, after 3 months at 37°C., the pH 6 and pH 7 saline formulated material (originally containing92 to 93% supercoiled plasmid DNA) contains 0% initial supercoiled, ˜36%open circle and ˜64% linear plasmid DNA.

[0138] To determine if the trends observed with saline versus PBSformulated probe material under accelerated conditions corresponds tomaterial under nonaccelerated storage conditions, a stability study wassetup. Plasmid DNA was formulated at low, intermediate and high doses inboth saline (2, 20 and 2200 mcg/mL plasmid) and PBS (2, 20, 1000 mcg/mLplasmid). Both solution pH and supercoiled content were monitored duringstorage at 2-8° C. and −70° C. As shown in Table 1, the PBS formulatedmaterial was more consistent in terms of pH and plasmid supercoiled DNAcontent during storage for up to 3 months.

[0139] To examine the effects of different buffer ions and pH on DNAstability several lots of plasmid DNA were formulated with severaldifferent buffer/pH combinations and incubated for 3 months at 37° C.Following the incubation, the percent of initial supercoiled DNA contentwas determined by agarose gel electrophoresis. The results shown inTable 6 indicate that the stability is highly variable from lot to lotof DNA, that acidic pH is detremental to stability and that formulationshaving a higher DNA concentration were more stable. The lot to lotvariability may have been due to varying trace metal TABLE 7 Table 7:Comparison of the effects of different buffer ions and pH on DNAstability, at 5, 24 and 37° C. Percent of initial supercoiled DNAremaining was determined by agarose gel electrophoresis. Percent ofinitial supercoiled DNA remaining. 1 month Condition Initial % SC 5° C.24° C. 37° C. A 97 90 86 43 B 97 94 89 40 C 95 92 47  0 D 94 89 66 16 E94 93 63 32 F 95 94 98 58 G 96 94 94 79

[0140] content. The results of Table 6 suggested that certain buffer/pHcombinations would require further investigation. The most promisingcombinations identified by this study were sodium bicarbonate (pH 7.2),PBS (pH 7.2-8.0) and sodium succinate (pH 7.2).

[0141] To address the question of whether the addition of a buffer tosaline improves the stability of the DNA, a study was initiated tocompare the effects of saline alone, with saline in combination withfour different buffers. For this study the DNA was formulated at 20mcg/mL and incubated at 37° C. for 3 months. The percent of initialsupercoiled DNA was determined by agarose gel electrophoresis. Theresults, shown in FIG. 7B, clearly indicate that the addition of eithera sodium bicarbonate, phosphate, Hepes or Tris buffer improved thestability of the DNA, compared to saline alone. The data also suggestedthat there are major differences in the stabilizing effects of thebuffer ions. Furthermore, the results suggest that the DNA stability isrelated to the maintenance of pH. As seen in FIG. 7B, the Tris/salinecombination and particularly saline alone had the least stabilizingeffect on the DNA, and did not maintain a constant pH over the 3 monthsof incubation. Therefore, one of the functions of the buffer, withrespect stabilizing DNA, is to maintain the pH in the neutral toslightly basic range. Although another function of the buffer is tostabilize the DNA against degradative pathways, these results indicatethat different buffers have different capacities to stabilize DNA, andfurther work was needed to identify the particular buffers having thegreatest stabilizing effect. In that regard, these data were the firstindication that sodium bicarbonate stabilizes DNA during storage.

[0142] The effects of additional buffer/pH combinations on DNA stabilityare shown in Table 7. In this study the DNA was formulated in variousbuffer/pH combinations and incubated for one month at 50, 24° and 37° C.Following the incubation the percent of initial supercoiled DNA wasdetermined by agarose gel electrophoresis. The results indicated thatthe best buffer/pH combination was Tricine at pH 7.5. However, severalother combinations provided stability above that of sodium phosphate atpH 7.5, including sodium succinate (pH 6.2), sodium malate (pH 6.2) andTris (pH 7.5). Additional studies designed to compare the most promisingbuffer/pH combinations again are in progress at 4°, 25°, 37° and 50° C.

[0143] To determine if the buffer ions in the DNA vaccine formulationwould affect the immune response, the induced immune response toInfluenza DNA vaccine, HA (Georgia/93) was measured at several doses ofDNA by measuring HI titer. The results shown in FIG. 9 indicate that theimmune response to the DNA vaccine formulated in PBS was superior to theresponse observed for DNA formuated in saline.

[0144] To determine the effects of pH on DNA stability over a broadrange of pH, a stability experiment was performed in 20 mMBis-Tris-Propane containing 150 mM NaCl, from pH 6.0 to 9.0. The resultsof the stability test are shown in FIG. 13, after 2 weeks of incubationat 50° C. (2 mcg/mL DNA). The results clearly indicate that the pH ofthe formulation greatly affects the stability of the DNA, and that theoptimum pH is ≧8.5. Since the stability of the DNA in the PBS control,at pH 7.1, was nearly equal to that of the DNA in Bis-Tris-Propane at pH7.5, the results suggest that the buffer type also influences thestability of the DNA at a given pH.

[0145] To address the effects of buffer type on DNA stability astability experiment was performed with DNA at 20 mcg/mL in sevendifferent formulations. Since previous experiments had shown that thepurity of the buffer influenced DNA stability, 10 mM succinate and 2%ethanol were added to each formulation to inhibit free radical oxidationof the DNA (data presented in examples 14 and 15 show that succinate andethanol each enhance DNA stability). The results (FIG. 14) indicate thatthe formulation that provided the highest DNA stability was also at thehighest pH used (pH 9). However, some buffer effects were also noted.The largest buffer effect was between glycine and Bis-Tris-Propane,where the data indicates that the glycine formulation at pH 9 wassignificantly superior to the Bis-Tris-Propane formulation at the samepH. In contrast, the Tris, Bicine and Tricine buffers at pH 8.2,provided nearly identical DNA stability out to 12 weeks. The data alsoindicate that the two phosphate formulations (at pH 7.7 and 8.0)provided the lowest DNA stability, but they were also the lowest pHformulations tested. Due to the low buffering capacity of phosphatebuffers above pH 8.0, the phosphate formulations were limited to pHvalues at or below 8.0. These results suggest that once free radicaloxidation of the DNA is controlled by the addition of chelators (seeexample 15 for data showing which chelators enhance DNA stability andunder what conditions) and free radical scavengers (see example 14 fordata showing the enhancement of DNA stability by ethanol) that thestability of the DNA is controlled primarily by the pH of theformulation.

EXAMPLE 12

[0146] Effect of light on DNA stability—Initially, the light sensitivityof the material was evaluated to determine if special handlingprocedures would be required or if amber vials would be necessary forlong term storage. The results of an experiment to measure lightsensitivity (FIG. 10) indicated that exposure of 100 mcg/mL plasmid DNAin saline to light accelerated the conversion of supercoiled DNA plasmidto the open circular form, as measured by agarose gel electrophoresis.The conversion to the open circular form was much more pronounced whenthe DNA was stored in clear glass vials as opposed to amber vials, asexpected. Although a 2-4 week exposure to light caused significantdegradation, no significant losses were observed over an eight hour day.Since amber vials have the potential to leach trace metal ions overtime, which catalyze free radical oxidation of the DNA (see below), apackaging solution to the light sensitivity of the plasmid is requiredfor long term storage.

[0147] To determine the effects of light on DNA stability informulations containing Fe⁺³, metal ion chelators and free radicalscavengers a stability study was performed over 9 weeks at 30° C. in thepresence and absence of visible light (fluorescent light at 2000 Lux).The DNA concentration was 20 mcg/mL and the formulation buffer was 10 mMsodium phosphate containing 150 mM NaCl at pH 8.0. The results, shown inFIGS. 27 and 28 below, indicated that light decreased the stability ofthe DNA significantly in the PBS control and in PBS containing 500 ppbFe⁺³ or PBS containing 0.5 mM EDTA and 500 ppb Fe⁺³. The results alsoindicated that the presence of 0.5 mM EDTA did not diminish thedetrimental effects of light on DNA stability in the presence of 500 ppbFe⁺³. The results in FIG. 16 show the effects of light on DNA stabilityin formulations containing EDTA and ethanol. The results indicated thatthe presence of EDTA and ethanol greatly stabilized the DNA, in thelight and dark samples, compared to the control formulation in FIG. 15.The results also indicated that the detrimental effects of light on DNAstability were greatly diminished by the presence of EDTA and ethanol,even in formulations containing 500 ppb Fe+3. Therefore, the resultssuggest that DNA vaccine formulations containing EDTA and ethanol wouldbe much less sensitive to the detrimental effects of light and tracemetal ions than formulations lacking either of these two stabilizers.

EXAMPLE 13

[0148] Effect of demetalation and deoxygenation on DNA stability: Methodof demetalating PBS. —To prepare demetalated PBS, 20.0 grams of Chelex100 resin (BIO-RAD Laboratories) was washed with 400 mL of USP waterusing vacuum filtration over a cellulose acetate membrane (0.22micrometer pore size). The washed resin was added to approximately 1liter of PBS and stirred slowly overnight at 2-8° C. by placing amagnetic stir bar in the one liter bottle containing the slurry andusing a magnetic stirrer set to its slowest stir rate. The following daythe resin was removed by vacuum sterile filtration using a celluloseacetate membrane (Corning, 0.22 micron pore size). Care was taken topre-wash the membrane with two 10 mL applications of the demetalatedslurry prior to collecting the final filtered product. This step wasperformed to ensure that any potential leaching of metal ions from themembrane would not contaminate the demetalated product.

[0149] Method of demetalating Plasmid DNA—To demetalate solutionscontaining plasmid DNA, the DNA was diluted with demetalated PBS to afinal volume of approximately 2 mL. The DNA was then applied to a 1 mLChelex 100 column, previously equilibrated by washing the column with 5mL of demetalated PBS. The demetalated DNA in the effulent wascollected, followed by the addition of 6 mL of additional demetalatedPBS to wash the remaining DNA off the column. The entire effluent wasthen diluted to a final volume of 12.0 mL with demetalated PBS andsterile filtered with a Millipore Millex-GV 25 mm syringe filter (0.22micron pore size). Our inital experiments involved the demetalation ofonly 48 micrograms of DNA with a 1 mL column, however, the capacity ofthe Chelex 100 resin will allow much larger quantities of DNA to bedemetalated using the same size column.

[0150] Method of deoxygenating PBS, and demetalated PBS. —To preparedeoxygenated (degassed) PBS or deoxygenated and demetalated PBS, 250 mLof the buffer was heated to boiling in a Pyrex bottle. The solution wasthen allowed to cool to room temperature under continuous heliumsparging, and capped.

[0151] Method of preparing deoxygenated, or deoxygenated and demetalatedplasmid DNA—To prepare deoxygenated DNA solutions for stability studies,Influenza vaccine DNA, HA (Georgia/93), was diluted into steriledeoxygenated PBS. The orignal stock Influenza vaccine DNA was notdeoxygenated because it was diluted over 1000-fold (from 2.55 mg/mL to2.0 mcg/mL). The deoxygenated DNA solution was then placed in sterileglass vials, and capped after flooding the headspace with filterednitrogen. To prepare deoxygenated and demetalated DNA solutions forstability studies, Influenza vaccine DNA, HA (Georgia/93), was firstdiluted into demetalated and deoxygenated PBS. This solution was thenapplied to a 1 mL Chelex 100 column to demetalate the DNA. The solutionwas then diluted with additional deoxygenated and demetalated PBS,filter sterilized and sparged with helium just prior to filling vials.The headspace of each vial was flooded with filtered nitrogen beforecapping.

[0152] Table 6 contains the results of an experiment to determine thestability of plasmid DNA in PBS (pH 7.2) and demetalated PBS. Theresults indicate an improvement of DNA stability in demetalated PBS overthe control condition, and suggest that the stability of a DNA vaccinewould be greatly enhanced if stored in a demetalated buffer.Furthermore, DNA stored in demetalated PBS was far more stable thanwould be predicted using the published rate constants for depurinationand β-elimination. Depurination and β-elimination are two sequentialchemical reactions in the process of breaking the phosphodiesterbackbone of the DNA, that occur in aqueous solution. In the first step,[H⁺] catalyzes the loss of the purine bases from DNA, leaving behind anapurinic site (AP site). In the second step of the hydrolysis, [OH⁻]catalyzes a β-elimination reaction, which breaks the bond between theoxygen atom bonded to the 3′ carbon of the deoxyribose, and the 3′carbon atom. Because depurination and β-elimination are natural procesesthat cannot be completely prevented from occuring, in aqueous TABLE 8Table 8: Effects of demetalation, deoxygenation and p-hydroxybenzylalcohol on DNA stability. Percent of initial supercoiled DNA remainingwas determined by agarose gel electrophoresis. % of Initial SupercoiledDNA Remaining Con- 25-Jan di- Initial 26-Jan 27-Jan 29-Jan 8-Feb 22-Feb7-Mar tion % SC 1 day 2 days 4 days 14 days 28 days 42 days C1 90 97 9382 45 0 0 C2 89 98 102 94 90 65 33 C3 89 104 101 94 68 21 4 C4 90 98 10298 94 20 37 C5 91 90 77 36 0 0 0 C6 92 99 95 82 21 0 0 C7 91 97 92 78 00 0

[0153] solution, one would expect that when all other sources of DNAdegradation are eliminated, that the natural rates of depurination andβ-elimination would define the stability of the DNA. The published rateconstant (k) and activation energy (Ea) for depurination at pH 7.4 (70°C., +10 mM MgCl₂) are 4.0×10⁻⁹ s⁻¹ and 31 kcal/mol, respectively (seeLindahl et al.,1972, Biochemistry 19: 3610-3618). Therefore, at 50° C.the depurination rate in PBS should be approximately 2.3×10⁻¹⁰ s⁻¹ inthe presence of magnesium, and 3.3×10⁻¹⁰ s⁻¹ in the absence ofmagnesium, based on the effects of magnesium at 70° C. The publishedrate constant (70° C., in the absence of magnesium) and Ea for theβ-elimination step are 2.4×10⁻⁵ s⁻¹ and 24.5 kcal/mol (see Lindahl etal., 1972, Biochemistry 19: 3610-3618). Therefore, at 50° C. the rateconstant for the β-elimination step should be approximately 2.6×10⁻⁶ S₁.Therefore, the rate of strand break (SB) formation would be equal to [APsites]k₂, where the k1 is the rate constant for depurination, k2 is therate constant for β-elimination and [AP sites] is the concentration ofapurinic sites in the DNA, as shown below.

Plasmid DNA - - - k₁ - - - >[AP sites] - - - k₂ - - - >Strand breaks(SB)

[0154] Therefore, to determine the rate of strand break formation at anypoint in time, after a period of incubation at 50° C.

[0155] Rate=6,600 purines (for IDV, HA, Georgia/93)×3.3×10⁻¹⁰ s⁻¹×k₂

[0156] Rate=2.2×10⁻⁶ s⁻¹×k₂

[0157] Rate=2.2×10⁻⁶ s⁻¹×2.6×10⁻⁶ s⁻¹

[0158] Rate=5.7×10⁻¹² s⁻²

[0159] Rate of SB formation at any time=5.7×10⁻¹² s⁻²×(time in seconds)

[0160] Then, the number of SB present at any time t is equal to theintegral of 5.7×10⁻¹² (t) over time from t=0 to t=time

[0161] Then, the number of SB present at any time t=½ (5.7×10⁻¹²) t²

[0162] Therefore, the number of SB present at any time t=2.85×10⁻¹² (t²)

[0163] If we use the published rate constants above to determine thenumber of strand breaks in the Influenza DNA vaccine after 28 days ofincubation at 50° C., we obtain 16.7 strand breaks per plasmid. A randomdistribution of 16.7 strand breaks per plasmid molecule in a populationof plasmids would produce a composition completely devoid of anysupercoiled DNA. However, the data in Table 6 indicate that in thedemetalated sample, 65% of the initial supercoiled DNA remained after 28days. These data suggest that demetalation greatly reduces the rate ofdepurination and/or β-elimination, in aqueous solution. It is not knownhow trace metal ions could affect the depurination or β-eliminationreactions to this degree.

[0164] The results in Table 8 also indicate that deoxygenation improvedDNA stability, presumably by reducing the production of oxygencontaining free radicals. The most stable formulation in this study wasthe demetalated, deoxygenated sample, having 37% of initial supercoiledDNA remaining after 42 days at 50° C.

[0165] The results of another study on the effects of demetalation(Table 9) also indicate that demetalation and deoxygenation of the PBSbuffer and the DNA greatly improves DNA stability at 50° C., over thenon-demetalated PBS control. In order to remove any residual metal ionsfrom the glass vials used for storage, some of the vials were washedwith a solution of PBS containing I mM EDTA, deionized water andautoclaved before use. A comparison of the DNA stability between washedand unwashed vials indicated that washing the vials did notsignificantly improve the stability of the DNA, under acceleratedconditions. However, washing the vials to reduce the surface metal ioncontent may improve the long term stability of the DNA vaccine.

[0166] To determine if the enhanced stability observed in demetalatedPBS requires the demetalation of the plasmid DNA prior to its additionto the demetalated PBS, a study was initiated to determine TABLE 9 Table9: Effects of demetalation, deoxygenation and ethanol on DNA stability.Percent of inital supercoiled DNA was determined by agarose gelelectrophoresis. % of Initial Supercoiled DNA remaining 6-Feb 13-Feb20-Feb 5-Mar 19-Mar 2-Apr Condition Initial % SC 7 days 14 days 28 days42 days 56 days C1 95 86 59 19 3 0 C2 97 95 85 52 44 14 C3 95 99 86 4753 9 C4 97 97 92 72 60 33 C5 95 93 89 63 58 29 C6 97 98 93 71 68 42

[0167] TABLE 10 Table 10: Effects of partial and complete demetalation,EDTA and ethanol on DNA stability. Percent of initial supercoiled DNAremaining was determined by agarose gel electrophoresis. % of InitialSupercoiled DNA remaining 20-Feb 27-Feb 5-Mar 19-Mar 2-Apr 16-AprCondition Initial % SC 7 days 14 days 28 days 42 days 56 days C1 95 7638 15 0 C2 95 90 74 55 43 C3 95 88 71 49 27 C4 94 83 41 14 0 C5 96 89 5625 12 C6 96 89 57 29 13 C7 94 100 91 78 63

[0168] TABLE 11 Table 11: Effects of removing any residual contaminatingnuclease activity, on DNA stability. Percent of initial supercoiled DNAremaining was determined by agarose gel electrophoresis. % of InitialSupercoiled DNA remaining 29-Feb 7-Mar 14-Mar 28-Mar 11-Apr 25-AprCondition Initial % SC 7 days 14 days 28 days 42 days 56 days C1 95 7850 19 C2 96 82 56 22 C3 95 70 32 13 C4 95 69 45 7 C5 95 82 56 20

[0169] the effect on stability of a 1000-fold dilution of thenon-demetalated plasmid DNA into demetalated PBS. The results (Table 10,conditions C1-C3) indicated a reduction in stability when the DNA wasnot demetalated, suggesting that it is necessary to demetalate the DNAand the PBS to obtain maximum stability.

[0170] In order to determine if the enhanced stability in thedemetalated conditions was due to the inactivation of residualcontaminating nuclease activity (caused by metal ion removal) in theDNA, we performed an experiment to determine the effects of removing ordenaturing any contaminating nuclease activity on DNA stability. Theresults (Table 11) indicated that the addition of 0.1% SDS did notsignificantly enhance stability over the PBS control. Furthermore,treatment of the DNA with 1 mcg of protease K for 1 hour at 24° C.,followed by removing the enzyme with a Micropure EZ membrane, did notenhance stability. The Micropure EZ membrance treatment alone, also didnot improve stability. In addition, we found that a phenol extractionand ethanol precipitation of the DNA had no effect on stability. Theseresults strongly suggest that the enhanced stability observed indemetalated PBS is not due to the inactivation of residual nucleaseactivity, but is instead due to the removal of metal ions capable ofcatalyzing free radical oxidation of the DNA.

[0171] To further examine the effects of demetalation on DNA stability aseries of DNA stability experiments were carried out using demetalatedbuffers with an improved batch binding demetalation procedure thatensures efficient removal of trace metal ions and no alteration of thebuffer pH. Buffers were demetalated by placing 5 g of Chelex resin in˜75 mL of the buffer to be demetalated. With the solution being stirredat a moderate rate on a magnetic stirrer, 1N HCl was added to the slurrydropwise, to adjust the pH to the desired pH of the buffer. Then,additional 1N HCl was added over the next 15-30 minutes until the pH ofthe slurry stabilized at the desired pH. When the pH had stabilized, theslurry was filtered and the washed and pH adjusted Chelex resincollected. The entire 5 grams of washed resin was then placed in 250 mLof the buffer to be demetalated (in a 250 mL capped bottle) and stirredslowly overnight, at 2-8° C. Then, the buffer was filter sterilizedusing a Corning vacuum filtration unit with a 0.22 mm cellulose acetatemembrane. Prior to collecting the filtered buffer the membrane of theCorning filtration unit was washed twice, with 5-10 mL of the slurryeach time, to remove any trace metal ions from the cellulose acetatemembrane and to wash the polystyrene container. The sterile demetalatedbuffer was stored at 2-8° C. until used.

[0172] To determine the effects of reagent purity and demetalation onDNA stability, plasmid DNA at 2 mcg/mL was incubated in PBS made withreagents from two different sources at pH 7.2 for 6 weeks at 50° C. Theresults (FIG. 15) indicated that demetalation of the formulation buffersignificantly enhanced DNA stability for PBS made with the B reagents,but had little effect on the stability of DNA in PBS made with the Areagents. These results suggest that trace metal ion impurities informulation buffer reagents can lead to DNA degradation during storage,and that demetalation of the less pure formulation buffers improves DNAstability.

[0173] To determine the effects of demetalation on DNA stability informulations of a higher pH, a stability study was performed over 6weeks at 50° C., with DNA at 2 mcg/mL in PBS and demetalated PBSadjusted to pH 8.0. The results, shown in FIG. 16, indicate thatdemetalation of the formulation buffer also improved DNA stability at pH8.0, in PBS.

[0174] To determine the effects of demetalation on DNA stability informulations containing free radical scavengers and metal ion chelatorsa DNA stability experiment was performed over 6 weeks at 50° C. withplasmid DNA at 2 mcg/mL. Two formulations were tested, each in thedemetalated and non-demetalated state. PBS was used as the buffer and 10mM sodium succinate as the chelator. Ethanol and glycerol were used asfree radical scavengers, each at 2% (v/v). The results, shown in FIG.17, indicate that demetalation improved DNA stability slightly in theformulation containing glycerol, but had no effect on DNA stability inthe ethanol formulation. These results suggest that the same level ofDNA stability can be achieved either by controlling free radicaloxidation with succinate and ethanol, or by removal of the trace metalions with demetalation. However, the addition of succinate and ethanolwould be expected to protect the DNA from any trace metal ionsintroduced during DNA vaccine formulation and filling. Althoughdemetalation would not protect the DNA from metal ions introduced afterthe demetalation procedure it would reduce the load of trace metal ionsin the formulation and may increase DNA stability during storage overmuch longer periods of time.

[0175] To examine the effects of demetalation on DNA stability in otherbuffer types, a DNA stability experiment was performed with plasmid DNAover 6 weeks at 50° C. with DNA at 2 mcg/mL in either a bicarbonate or aborate containing buffer. The results, shown in FIG. 18 below, indicatethat demetalation greatly increased DNA stability in each of theformulations. Therefore, the data suggests that demetalation is aneffective way to enhance DNA stability in a variety of formulationbuffers over a wide range of pH.

[0176] To determine if demetalation of the formulation buffer for a DNAvaccine enhances DNA stability at lower temperatures and over muchlonger periods of storage, a DNA stability experiment was performed over9 months at 30° C. with DNA at 2 mcg/mL, in two formulations. Theresults, shown in FIG. 19, indicate that demetalation of the PBS andbicarbonate containing formulations significantly improved DNAstability. These results suggest that the enhancement of DNA stabilityby demetalation is effective over a wide range of temperatures and timein storage.

EXAMPLE 14

[0177] Effect of free radical scavengers on DNA stability—It is nowwidely recognized that one mechanism of DNA degradation involves freeradical oxidation by molecules such as hydroxyl radicals. One way toprevent or minimize the amount of DNA damage by free radicals is to adda free radical scavenger to the solution. These molecules serve thepurpose of protecting the DNA, by competing with the DNA for the freeradicals. Since the scavengers are compounds selected to be highlyreactive towards free radicals and are often present at higherconcentrations than the reactive part of the DNA (usually thedeoxyribose sugar), they effectively protect the DNA from damage.

[0178] To determine if free radical oxidation was occuring duringstorage and to text the effectiveness of several free radicalscavengers, Influenza DNA vaccine, HA (Georgia/93) was formulated insaline containing 4% (w/v) mannitol, 4% (v/v) glycerol, 5 mM methionineor 10 mM sodium azide (known free radical scavengers) and incubated at37° C. for three months. The DNA was subjected to agarose gelelectrophoresis at three timepoints to determine the percent of initialsupercoiled DNA remaining. The results in FIG. 11, indicate that insaline, glycerol, methione and sodium azide stabilized the DNA, comparedto the saline control. These early results suggested that free radicaloxidation was occuring during storage, and that three different freeradical scavengers were effective stabilizers of the DNA.

[0179] The results of another study designed to examine the effects ofthe free radical scavenger dimethyl sulfoxide (DMSO), and the TABLE 12Table 12: Effects of reducing agents, Desferal and dimethyl sulfoxide onDNA stability at 5, 24 and 37° C. The percent of initial supercoiled DNAremaining was determined by agarose gel electrophoresis. Percent ofinitial supercoiled DNA remaining. 30-Aug 1 Month 3 Month condition %InitialSC 5C 25C 37C 5C 24C 37C A 70 0 0 0 0 0 0 B 48 0 0 0 0 0 0 C 61 00 0 0 0 0 D 100 100 96 0 100 44 0 E 100 100 93 0 100 12 0 F 100 100 10091 100 100 58 G 100 99 99 89 100 91 37

[0180] TABLE 13 Table 13: Effect of free radical scavengers on DNAstability at 5, 24 and 37° C. Percent of initial supercoiled DNAremaining was determined by agarose gel electrophoresis. Percent ofinitial supercoiled DNA remaining. 29-Sep 1 Month 3 Month ConditionIntial % SC 5C 24C 37C 5C 24C 37C A 100 100 100 90 100 95 46 B 100 100100 84 100 90 46 C 100 100 97 77 99 83 30 D 100 77 45 5 63 26 0 E 97 100100 51 99 83 0 F 100 100 100 55 98 78 0 G 100 100 100 94 99 90 54

[0181] reducing agents ascorbic acid, sodium metabisulfite, sodiumsulfite and thioglycerol on DNA stability, is shown in Table 12. In thisexample, DNA was formulated at 20 mcg/mL in PBS (pH 7.2) and incubatedat 5, 24 and 37° C. The results indicate a dramatic destruction of theDNA in the presence of all of the reducing agents, and an enhancement ofstability with 0.2% (v/v) DMSO, compared with the PBS control. Theresults of this experiment are consistent with those of FIG. 11, thatmost of the non-reducing free radical scavengers tested, have stabilizedthe DNA.

[0182] A re-examination of free radical scavengers was then performed toexamine the effects of 10% (v/v) glycerol, 10 mM methionine and 2% (v/v)ethanol on DNA stability. In this experiment the DNA was formulated at20 mcg/mL in PBS (pH 7.2) and incubated at 5, 24 and 37° C. The results,shown in Table 13, indicated that 2% ethanol in PBS was the mosteffective stabilizer. However, glyerol and methionine also showed somestabilizing effect. Another result from this study was that the PBSformulations were much more stable than the saline formulations,presumably because the pH tended to drift down in the saline formuationsover time, causing an increase in the rate of degradation.

[0183] The results of an experiment to examine the effects free radicalscavengers pentoxifylline, tert-butylhydroquine and p-aminobenzoic acidon DNA stability are shown in Table 14. In this study the IDV, HA(Georgia/93), was formulated at 2.0 mcg/mL in PBS (pH 7.2) in thepresence of a 10 mM concentration of the scavenger. The samples for thisstudy were incubated at 50° C. and examined for supercoiled DNA contentby agarose gel electrophoresis. The results indicated that none of thescavengers tested improved the stability of the DNA, and in fact theygreatly accelerated the degradation of the DNA. It is not clear whythese scavengers accelerated the degradation of the DNA, while ethanol,DMSO, glycerol and methionine provided an enhancement in stability.

[0184] The ability of ethanol to stabilize the DNA was also examined indemetalated PBS, as well as in demetalated and deoxygenated PBS. Forthese two studies the DNA was formulated at 2.0 mcg/mL and incubated at50° C. The results, shown in Tables 9 and 10, indicate that 5% (v/v)ethanol stabilized the DNA in PBS, and in either of the demetalatedformulations.

[0185] Recent results from the first time point (1 month) of a long-termstability study also indicate that 5% ethanol stabilizes the DNA indemetalated PBS at 37° C., in formulations containing 2.0 mcg/mL DNA.The one month time point indicated that the PBS control had 93% of theinitial supercoiled DNA remaining, while the demetalated sample had96.1% and the demetalated sample containing 5% ethanol had 100%.

[0186] To examine the effects of free radical scavengers on DNAstability, two DNA stability experiments were performed over 8 weeks at50° C. with DNA at 20 mcg/mL in PBS at pH 7.2 and pH 8.0. Since ethanolis an effective free radical scavenger approved for human use, ethanolwas tested as the scavenger at 2% (v/v) in the presence and absence ofEDTA. The results of the first experiment (at pH 7.2) are shown in FIG.20 below. The results indicate that ethanol alone enhanced DNAstability, while EDTA alone decreased DNA stability. The combination ofethanol and EDTA provided a large increase in DNA stability up to 4weeks, but only a small increase in stability by week 8. These resultssuggest that ethanol is a more effective scavenger of free radicals inthe presence of EDTA, than in its absence. Moreover, the results suggestthat EDTA alone decreases DNA stability in the absence of ethanol, butincreases DNA stability in the presence of ethanol. These resultsstrongly suggest that ethanol is a more effective scavenger in thepresence of EDTA because EDTA removes metal ions bound to DNA, therebyallowing the generation of hydroxyl radicals in the bulk solution, asopposed to the generation of radicals by iron bound to the DNA. Theproduction of hydroxyl radicals in the bulk solution would allow ethanolmolecules more time to scavenge the radicals since the mean free path ofthe radical would be longer before its interaction with the DNA.Hydroxyl radicals generated by iron molecules bound to the DNA would bein very close proximity to the DNA. Therefore, the ability of ethanol toscavenge radicals produced on the “surface” of the DNA would greatlydiminished. These results also suggest that chelators other than EDTAmay be effective DNA stabilizers, provided that the chelator is able toremove metal ions (iron and copper) already bound to the DNA.

[0187] The results of the second stability study, at pH 8.0 are shownbelow in FIG. 21. The data from this experiment clearly show that theDNA stabilizing effects of ethanol and EDTA/EtOH are greater at pH 8.0than at pH 7.2.

[0188] To determine whether the combination of succinate and ethanolwould provide the same degree of DNA stabilization as observed with theEDTA/EtOH combination, and to determine whether either of thesecombinations would protect DNA from the presence of Fe⁺³, a stabilityexperiment was performed over 6 weeks at 50° C. in 10 mM sodiumphosphate buffer containing 150 mM NaCl at pH 8.0 with 20 mcg/mL DNA.The results indicated that the succinate/EtOH combination did notprovide the same degree of DNA stabilization as EDTA/EtOH. However, thecombination of succinate and ethanol did provide nearly completeprotection from the enhanced free radical generation produced by theaddition of 500 ppb Fe⁺³. The results, shown in FIG. 22, also indicatedthat the EDTA/EtOH combination provided the best DNA stability overalland provided complete protection from the addition of 500 ppb Fe⁺³.These results suggest that the specific combination of EDTA and ethanolprovides greatly enhanced DNA stability, and that the same degree of DNAstability cannot be achieved by using the combination of succinate andethanol.

EXAMPLE 15

[0189] Effect of metal ion chelators on DNA stability—In preliminarystudies designed to examine the effects of buffer ions, pH and salt onDNA stability, we also examined the effect of adding 1 mM and 10 mM EDTAto DNA formulated in PBS (pH 7.2). For these early experiments theplasmid DNA was formulated at 100 mcg/mL and incubated at 60° C. for 48hours. The supercoiled DNA content was then determined by agarose gelelectrophoresis. The results, shown in Table 5, indicated that EDTA hadno effect on the stability of the DNA. Suggesting, at this early time,that trace metal ions were not required for the DNA degradation processwe were observing.

[0190] Other experiments have also included an examination of theeffects of EDTA on DNA stability. The results shown in Table 12, forexample, indicated that 0.5 mM EDTA had no significant effect on thestability of DNA formulated at 2.0 mcg/mL in PBS (pH 7.2), or in PBScontaining 5% (v/v) ethanol, when incubated at 50° C.

[0191] An early experiment designed to examine the effect of the ironchelator Desferal (at 1 mM) on DNA stability, indicated an enhanced rateof degradation. In this study the DNA was formulated in PBS (pH 7.2) at20 mcg/mL and incubated at 5, 25 and 37° C. The reason for the enhancedrate of degradation is not clear.

[0192] Later experiments also confirmed the detrimental effect ofDesferal on DNA stability. The results shown in Table 15, for example,show that 0.5 mM Desferal caused a rapid degradation of the DNA in PBS,even if the PBS was treated with Desferal before mixing it with the DNA.This experiment also showed that treatment of the DNA with 1.0 mMDesferal, overnight, prior to diluting it in demetalated PBS, stillresulted in a rapid degradation of the DNA. For these latest experimentswith Desferal, the DNA was formulated in PBS at 2.0 mcg/mL and incubatedat 50° C. TABLE 14 Table 14: Effects of metal ion chelators and freeradical scavengers on DNA stability. Percent of initial supercoiled DNAremaining was determined by agarose gel electrophoresis. % of InitialSupercoiled DNA remaining 6-Mar 13-Mar 20-Mar 3-Apr 17-Apr 1-MayConditions Initial % SC 7 days 14 days 28 days 42 days 56 days C1 97 7753 9 C2 98 85 69 34 C3 95 68 43 12 C4 97 65 29 0 C5 97 83 64 27 C6 97 7847 5 C7 19 0 0 0 C8 98 17 0 0

[0193] TABLE 15 Table 15: Effects of Desferal, a metal ion chelator, onthe stability of DNA. Percent of initial supercoiled DNA remaining wasdetermined by agarose gel electrophoresis. % of Initial Supercoiled DNAremaining 28-Feb 6-Mar 13-Mar 27-Mar 10-Apr 24-Apr Condition Initial %SC 7 days 14 days 28 days 42 days 56 days C1 97 82 58 17 C2 97 11 9 0 C398 86 68 22 C4 97 30 4 0 C5 97 93 87 73 C6 98 92 87 68 C7 96 94 88 69 C897 92 86 62 C9 96 24 0 0 C10 97 19 0 0

[0194] The results of an experiment to examine the effects of some wellcharacterized metal ion chelators, on DNA stability, are shown in Table14. For these studies the DNA was formulated at 2.0 mcg/mL in PBS (pH7.2) and incubated at 50° C. Four different chelators were tested, eachat 0.5 mM. The results indicated that inositol hexaphosphate (IHP) andtripolyphosphate (TPP) improved the stability of the DNA. However,ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) anddiethylenetriaminepenta-acetic acid (DTPA) did not enhance stability.These results suggest that IHP and TPP may be useful to furtherstabilize the DNA in demetalated PBS, and in demetalated PBS containingethanol as a free radical scavenger. Although the stabilizing effect ofIHP and TPP are consistent with the enhancement of stability withdemetalation, and the stabilizing effects of ethanol (based on themechanism of DNA degradation being a metal ion catalyzed, free radicaloxidation) it is not clear why other metal ion chelators do notstabilize the DNA. The published literature (see J. Biol. Chem. 259,3620-3624; 1984) suggests that IHP, EDDHA, DTPA and Desferal lack a freecoordination site on the metal, when coordinated to iron. Therefore, itwas reported that these four chelators do not produce hydroxyl radicalswhen complexed to iron, as EDTA does. With this understanding of thechemistry, it is difficult to explain the differential effects of thesechelators on DNA stability. However, our results do suggest thatchelators containing multiple phosphate ligands may be the mosteffective chelators to protect DNA from metal ion catalyzed oxidation.Furthermore, additional chelators with multiple phosphate ligands wouldinclude the various salt forms of polyphosphoric acid.

[0195] The results of our most recent studies to examine differentbuffers in the demetalated state (see Example 16) also suggests thatspecific metal ion chelators are important for stabilizing DNA duringstorage. Since demetalated PBS containing succinate or malate wassuperior to demetalated PBS alone, the stabilizing effect is likely tobe due to the binding of metal ions by the succinate and malate anions.However, our data also indicate that citrate, a commonly used bufferwith a higher affinity for metal ions than succinate, did not provideany stabilization of the DNA. Moreover, EDTA, Desferal, inositolhexaphosphate, EDDHA and DTPA all have very high affinities for metalions, but they do not stabilize the DNA during storage. Therefore, theability of the metal ion chelator to stabilize DNA formulations is notrelated to the binding affinity of the chelator for metal ions. Thisconclusion suggests that the identification of effective chelators willrequire an empirical screening of molecules with multiple phosphateligands, or with a chemical resemblance to succinic or malic acid.

[0196] To examine the effects of metal ion chelators on DNA stability, aseries of DNA stability experiments were performed. The purpose of thefirst experiment was to determine the effects of several differentchelators on DNA stability in PBS at pH 8.0 in the absence of ethanol.The experiment was carried out over 2 weeks at 50° C. using 20 mcg/mLDNA. The results, shown in FIG. 23, indicate that only NTA(nitrilotriacetic acid) and DTPA (diethylenetriaminepentaacetic acid)enhance DNA stability in the absence of ethanol. The data in FIG. 23 isconsistent with the previous results shown in FIGS. 21 and 22, in thatEDTA decreased DNA stability in the absence of ethanol. These resultssuggest that, among these chelators, only DTPA is an effective DNAstabilizer in the absence of ethanol.

[0197] To examine the ability of metal ion chelators to enhance DNAstability in the presence of ethanol an experiment was performed using 5different chelators over 12 weeks at 50° C. in PBS at pH 8.0. Theresults, shown in FIG. 24, indicated that only DTPA and EDTAsignificantly increased DNA stability over the PBS control containing 1%EtOH. However, 200 mM DTPA also enhanced DNA stability by an equivalentamount, in the presence and absence of ethanol. These results areconsistent with a published report (see Graf et al. 1984, J. Biol. Chem.259: 3620-3624) that iron-DTPA complexes do not support the generationof hydroxyl radicals, and therefore one would expect that ethanol wouldnot be needed as a scavenger.

[0198] To determine the optimum concentration of EDTA for stabilizingplasmid DNA in PBS containing 1% EtOH at pH 8.0, a stability experimentwas performed over 6 weeks at 50° C. using 20 mcg/mL DNA. The results,shown in FIG. 25, indicate that 10 μM EDTA provided the same stabilityenhancement as 500 μM EDTA, under these conditions. These resultssuggest that EDTA is enhancing the stability of DNA by binding lowconcentrations of trace metal ions present in the formulation buffer, orin the DNA preparations. Based on these data, an increase in the DNAconcentration from 20 mcg/mL to 1.0 mg/mL may only require an increasein the EDTA concentration from 10 μM to 500 μM. Therefore, the use ofEDTA and ethanol to stabilize DNA vaccine formulations should notrequire greater than 1 mM EDTA, even with DNA vaccines with DNAconcentrations of 2.0 mg/mL.

EXAMPLE 16

[0199] Effect of demetaled buffers on DNA stability—Our initial studieson the effects of buffer ions were performed with buffers that were notdemetalated. However, because the trace metal ion content of the buffersprobably varied, depending on the purity of the buffer, the stability ofthe DNA in non-demetalated buffers may be largely determined by thetrace metal ion content. Therefore, it may be necessary to compare theeffects of demetalated buffers, to determine the true influence of thebuffer ions on DNA stability.

[0200] To examine the effects of different buffer ions on stability,four different demetalated buffers were prepared. The control buffer wasdemetalated PBS at pH 7.2. The other buffers tested were demetalated PBScontaining 10 mM sodium succinate (pH 7.2), demetalated PBS containing10 mM sodium malate (pH 7.2), demetalated sodium bicarbonate (10 mM)containing 150 mM NaCl, 10 mM Tris-Cl, 150 mM NaCl (pH 7.8) and 10 mMTricine, 150 mM NaCl (pH 7.8). The Tris and Tricine buffers were notdemetalated because the buffering ion is cationic and binds to theChelex 100 column. For this study of demetalated buffers the InfluenzaDNA vaccine was formulated at 20.0 mcg/mL and incubated at 50° C. Theresults of the first time point (two weeks) indicated that demetalatedPBS control had 75% of the initial supercoiled DNA remaining, while thesuccinate, malate, sodium bicarbonate, Tris and Tricine buffers had 98%,94%, 100%, 31% and 65% of the initial supercoiled DNA remaining. Theseresults indicate that the demetalated buffers containing succinate andmalate were superior to demetalated PBS, and that demetalated sodiumbicarbonate containing 150 mM NaCl was the most stable formulation.Although it is not clear why sodium bicarbonate is superior to PBS, thestabilizing effect of PBS containing succinate and malate is likely tobe due to ability of these compounds to chelate metal ions. Becausesuccinate and malate have a safe record of use in pharmaceuticalproducts, and can be used at relatively high concentrations, thesecompounds may be the most effective way to chelate trace metal ions, andthus stabilize DNA.

EXAMPLE 17

[0201] Effect of lyophilization on DNA stability—DNA is susceptible to anumber of different degradative precesses in aqueous solution, includingfree radical oxidation, depurination and b-elimination reactions. Oneapproach to minimize the rate of these precesses would be to lyophilizethe DNA, thus lowering the water content and molecular mobility. Todetermine the effects of lyophilization on DNA stability during storage,six different lyophilized formulations were prepared. The stability ofthe DNA in the lyophilzed samples was compared to a liquid PBSformulation at 20 mcg/mL, after a one month incubation at 37° C., byagarose gel electrophoresis. To prepare the lyophilized samples,Influenza DNA vaccine was formulated at 20 mcg/mL with the appropriatestabilizers and 0.8 mL of solution was placed into 3 mL glass vials. Thesamples were then placed in the lyophilizer, and frozen during coolingof the shelf to approximately −45° C. over a period of 4 hours. A vacuumwas then applied (20 mTorr), while the shelf temperature was maintainedbetween −30° and −20° C. for 26 hours. The temperature of the shelf wasthen raised to 0° C. for 2 hours, then to a temperature of 25° C. for 6hours at a specific rate. The vacuum was then released and the vialswere stoppered under a nitrogen atmosphere.

[0202] The results of the lyophilization study, shown in FIG. 12,indicated that the DNA in four of the six lyophilized formulations wasmuch more stable than the DNA in the liquid PBS control, suggesting thatlyophilization will be a very effective way of stabilizing DNA vaccines.Interestingly, DNA vaccine formulations containing amorphous sugars suchas sucrose and lactose, greatly stabilize the DNA, while crystallinesugars such as mannitol do not enhance DNA stability compared to thesolution control (in PBS). Studies to be initiated soon include the useof demetalated buffers to prepare the lyophilized DNA samples. Sincemetal ions are very detremental to DNA stability in the liquid state,demetalated and lyophilized DNA formulations may provide greatlyenhanced stability over liquid formulations.

[0203] To determine the stability of lyophilized DNA vaccines and todetermine the effects of demetalating the formulation buffer on thestability of lyophilized DNA, a stability study was performed on thelyophilized samples over 4 months at 50° C. Three formulations weretested, each in the demetalated and non-demetalated state. The DNAconcentration prior to the lyophilization was 20 mcg/mL. Thelyophilization conditions were the same as described in this Examplesection. After 4 months of incubation the samples were resuspended insterile water and analyzed by agarose gel electrophoresis to determinethe % SC, OC and linear DNA. The results, shown in FIG. 26, indicatedthat the stability of the best lyophilized formulation exceeded that ofthe liquid formulations. However, the predicted 4 month stability of thebest liquid formulation exceeded that of lyophilized formulations 1, 5and 6. The 4 month stability data for the liquid formulations were basedon extrapolations of stability data from 3 months (formulations 8 & 9)or 6 weeks (formulation 7) at 50° C. and 20 mcg/mL DNA. The results alsoindicated that demetalation improved the stability of the lyophilizedDNA in formulations 1 and 2, but had little effect on the % SC DNA inthe other lyophilized formulations. These results suggest thatlyophilization is an effective way to stabilize DNA vaccines and thatdemetalation of the formulation buffer improves the stability oflyophilized DNA in some formulations.

EXAMPLE 18 EFFECT OF EDTA AND ETHANOL ON THE DEPURINATION ANDβ-ELIMINATION RATE CONSTANTS

[0204] The results of DNA stability studies disclosed herein suggestthat in the absence of free radical scavengers and metal ion chelatorsfree radical oxidation is the major mechanism of DNA degradation instorage. The data also supports the hypothesis that trace metal ions anddissolved oxygen in formulation buffers cause free radical oxidation ofthe DNA. Based on this hypothesis, the major mechanisms of DNAdegradation in formulations where free radical oxidation is effectivelycontrolled (by EDTA and ethanol) would be the processes of depurinationand β-elimination, which occurs to DNA in aqueous solutions (Lindahl etal., 1972, Biochemistry 11: 3610-3618). If depurination andβ-elimination are the predominant mechanisms of degradation for DNA instorage, then it is possible to accurately predict the % SC DNA overtime, using the published values of the depurination and β-eliminationrate constants and the activation energies (E_(a)) for these reactions.However, the results of several DNA stability studies involving DNAvaccine formulations containing ethanol and EDTA/EtOH have suggestedthat the DNA in these formulations is much more stable than one wouldpredict, based on the published rate constants. The reason for thehigher than expected DNA stability in formulations containing ethanol islikely to be due to the much lower levels of free radical oxidation.This hypothesis is consistent with known art disclosing ethanol as aneffective scavenger of hydroxyl radicals. Therefore, to determine theinherent stability of DNA in aqueous solution a stability study wasinitiated with DNA at 100 mcg/mL in PBS containing 1% EtOH and 0.5 mMEDTA at pH 7.4. To further control free radical oxidation of the DNA,the solutions were placed in sealed glass ampules. The ampules wereincubated at 40, 50, 60 and 80° C. After various periods of time ampuleswere removed from the incubator and the solutions assayed for % SC DNA,in the presence and absence of E. coli Exonuclease III, to determine thenumber of AP (apurinic) sites per plasmid and to determine the rateconstants for depurination (k₁) and β-elimination (k₂). A description ofthe AP site assay and the method for determining the depurination (k₁)and β-elimination (k₂) rate constants are recorded below.

[0205] Description of the AP Assay—The assay is based on the conversionof supercoiled plasmid DNA (containing AP sites) to the open-circleform, after treatment with Exonuclease III. Exonuclease III from E. colihas an associated AP-endonuclease activity that will cleave the DNAbackbone at AP sites, leaving DNA that lacks AP sites fully supercoiled.Since supercoiled plasmid DNA having only a single AP site per plasmidis completely converted to open-circle DNA by Exo III, the assay issensitive enough to detect the presence of AP sites when only 5 to 10%of the DNA molecules contain an AP site.

[0206] The assay is performed by incubating 100 ng of plasmid DNA withand without 0.25 units of Exo III for 30 minutes at 37° C., in a totalvolume of 35 mL. An aliquot of the DNA (18 ng) is then subjected toagarose gel electrophoresis and ethidium bromide staining. The negativeof the gel photograph is then scanned and the results are compared tosupercoiled, open-circle and linear standards that are applied to thesame gel, to allow quantitation of each form of DNA. To determine thenumber of AP sites in a sample of DNA, we use the data for percentsupercoiled DNA before and after treatment with Exo III. For example, ifa sample of DNA was 90% supercoiled before treatment and 50% supercoiledafter treatment, the calculation of the number of AP sites would be asfollows. First, based on past work, we assume that the rate ofdepurination is independent of DNA sequence and that the introduction ofAP sites follows a Poisson distribution. Then, the equation is used thatdescribes the number of strand breaks in a population of DNA molecules(SB) as a function of the fraction of DNA that is supercoiled (f₁).

SB=−ln f₁

[0207] For DNA that is 90% supercoiled, the number of strand breaks perplasmid, on average, is:

SB=−ln (0.90) or 0.105

[0208] For DNA that is 50% supercoiled:

SB=−ln (0.50) or 0.693

[0209] Therefore, the difference between 0.693 and 0.105 (0.588) is thenumber of strand breaks introduced by cleaving the DNA at the AP sitesand is therefore equal to the number of AP sites in the DNA.

[0210] Method for determining the depurination (k₁) and β-elimination(k₂) rate constants—To determine the depurination (k₁) and β-elimination(k₂) rate constants it is first necessary to derive an equation toestablish the mathematical relationship between these rate constants andsome DNA stability parameter that is easily measured. In this case wehave derived an equation that establishes the relationship between thenumber of strand breaks per plasmid (SB) and the number of AP sites perplasmid, with k₁ and k₂. Since we have already described therelationship between SB and the % SC DNA (above), we may then use DNAstability data (measuring % SC DNA over time) and the AP site assay todetermine k₁ and k₂, in DNA vaccine formulations containing EDTA andethanol.

[0211] In a population of molecules we can begin with the assumptionthat the number of strand breaks per plasmid (SB) at any point in time,is equal to the total number of AP sites produced up to that time (TAP)minus the number of AP sites remaining, per plasmid (AP). To simplifythe calculations we will also assume that the starting DNA contains noAP sites or strand breaks at time zero. Then,

SB=TAP−AP

[0212] Since TAP=k₁ (PB)t, where k₁ is the depurination rate constant,PB=number of purine bases and t=time. Then,

SB=k ₁(PB)t−AP

[0213] However, AP must be expressed in terms of k₁, k₂ and PB.Therefore, the rate of change of AP is set to equal to the rate ofproduction of AP sites, minus the rate of conversion to strand breaks.Then solve for AP by integration.

dAP/dt=k ₁(PB)−k ₂(AP)

dAP=k ₁(PB)dt−k ₂(AP)dt

∫dAP=∫k ₁(PB)dt−∫k ₂(AP)dt

[0214] Assuming that AP initial is zero, then;

∫k ₂(AP)dt=k ₂(AP)t

and; AP=k ₁(PB)t−k ₂(AP)t

[0215] Then, solving for AP;

AP=k ₁(PB)t/1+k ₂ t

[0216] Now, substituting this form of AP into our equation for SB, anequation is obtained indicating the number strand breaks per plasmid atany time.

SB=k ₁(PB)t−[k ₁(PB)t/1+k ₂ t]

[0217] Or, SB may be expressed in terms of AP.

[0218] Since, ∫k₂ (AP)dt=k₂ (AP) t, and ∫k₂ (AP)dt=SB

[0219] Then, SB=k₂ (AP)t.

[0220] Results of k₁ and k₂ Determinations—Using the above equations,determined k₁ and k₂ were determined for a DNA vaccine formulationcontaining EDTA and ethanol at 40, 50, 60 and 80° C. The results areshown in Table 16. Table 16 also shows a comparison of the measured k₁and k₂ to the published k₁ and k₂ values at the same pH and temperature.At 50° C., k₁ in this formulation is nearly 7-fold less than thepublished value, determined at the same pH and temperature. The resultsclearly indicate that the values of k₁ and k₂ in this formulation aremuch smaller than the published rate constants (Lindahl et al., 1972,Biochemistry 11: 3610-3618). This conclusion suggests that if freeradical oxidation of DNA is effectively controlled, that the DNA is morestable than one would predict, based on the published rate constants fordepurination and β-elimination. Moreover, the results suggest that thepublished rate constants are erroneously high, due to uncontrolled freeradical oxidation. TABLE 16 Measurements of k₁ and k₂ Using ExonucleaseIII*. Temperature mk₁ pk₁/mk₁ mk₂ pk₂/mk₂ 40 C. 1.38E−11 5.1 3.97E−07 250 C. 4.91E−11 6.8 1.44E−06 1.8 60 C. 2.73E−10 5.3 4.67E−06 1.7 80 C.2.15E−09 9.8 3.83E−05 1.7

[0221] To determine the activation energies for depurination (k₁) andβ-elimination (k₂) in the presence of EDTA/EtOH, and to determine if themechanism of degradation of DNA in PBS containing EDTA/EtOH ispredominately due to depurination and β-elimination, the above data wereused to make Arrhenius plots. The results are shown in FIGS. 29 and 30.The results indicate that the activation energies for depurination andβ-elimination are 28.4 and 25.2 kcal/mol, respectively. These valuesagree extremely well with the published values of 31±2 kcal/mol and 28kcal/mol for depurination (Lindahl et al., 1972, Biochemistry 11:3610-3618; Greer and Zamenhof, 1962, J. Mol. Biol. 4: 123), and 24.5kcal/mol for β-elimination (Lindahl and Andersson, 1972, Biochemistry11: 3618). These data suggest that the major mechanisms of DNAdegradation in PBS containing EDTA/EtOH are depurination andβ-elimination, and that the mechanism of these reactions is not alteredby the presence of EDTA and ethanol. These data also allow prediction ofthe percent supercoiled DNA remaining for DNA stability studiesperformed at other temperatures (at pH 7.4), provided that theformulation effectively controls for free radical oxidation.

[0222] To show that the stability of DNA in a DNA vaccine formulationcontaining 100 mM EDTA and 1% ethanol (at pH 7.4) is much greater thanpredicted by the published rate constants, the DNA stability data isplotted below (FIG. 31), along with two stability predictions. Theresults clearly indicate that the DNA in this formulation is much morestable than the published rate constants would suggest. Moreover, theresults indicate that the experimentally determined rate constants allowa much better prediction of DNA stability than does the use of thepublished rate constants.

[0223] Using the experimentally determined rate constants (k₁ and k₂)and the derived relationships between the rate constants and % SC DNA,it is possible to predict the formulation pH that would be required toachieve long-term stability at room temperature. These predictions (seeFIG. 32) suggest that the pH of a DNA vaccine formulation (containingEDTA/EtOH) would need to be approximately 8.0, in order to maintaingreater than 50% SC DNA for two years at 30° C., in a glass ampule.

[0224] These results show that EDTA/EtOH effectively controls freeradical oxidation in DNA vaccine formulations, and thereby enhances DNAstability to levels that would not be expected, based on the use of thepublished rate constants for depurination and β-elimination.

What is claimed is:
 1. A method of stabilizing highly purified DNAcomprising: a) isolating and purifying DNA to produce the highlypurified DNA; and b) optionally, removing metal ions from the purifiedDNA; and b) introducing the purified DNA into a solution to form astabilized DNA formulation, the solution being substantially free ofmetal ions.
 2. The method of claim 1 wherein the solution contains anonreducing free radical scavenging agent.
 3. The method of claim 2wherein the nonreducing scavenging agent is selected from the groupconsisting of ethyl alcohol, glycerol, methionine, dimethyl sulfoxide,and combinations thereof.
 4. The method of claim 1 wherein the solutioncontains a salt.
 5. The method of claim 2 wherein the solution containsa salt.
 6. The method of claim 3 wherein the solution contains a salt.7. The method of claim 1 further comprising the storage of thestabilized DNA formulation in the absence of light.
 8. The method ofclaim 2 further comprising the storage of the stabilized DNA formulationin the absence of light.
 9. The method of claim 4 further comprising thestorage of the stabilized DNA formulation in the absence of light.
 10. Astabilized DNA formulation comprising purified DNA, a nonreducing freeradical scavenging agent, a salt and a buffer.
 11. The formulation ofclaim 10 wherein the nonreducing free radical scavenging agent isselected from ethanol, glycerol, methionine, dimethyl sulfoxide, andcombinations thereof.
 12. The formulation of claim 11 wherein the saltis selected from NaCl, KCl, LiCl and combinations thereof.
 13. Theformulation of claim 12 wherein the buffer is selected from Tris-HCl,glycine, sodium phosphate, potassium phosphate, lithium phosphate,sodium succinate, potassium succinate, lithium succinate, sodium malate,potassium malate, lithium malate, sodium bicarbonate, potassiumbicarbonate, lithium bicarbonate and combinations thereof.
 14. Themethod of claim 1 wherein the purified DNA is suitable for humanclinical use.
 15. The method of claim 1 wherein the purified DNA isselected from influenza virus DNA, hepatitis A virus DNA, hepatitis Bvirus DNA, hepatitis C virus DNA, human papillomavirus DNA, DNA fromMycobacterium tuberculosis, human immunodeficiency virus DNA, varicellazoster virus DNA, herpes virus DNA, measles virus DNA, rotavirus DNA,mumps virus DNA, rubella virus DNA and combinations thereof.
 16. Amethod of stabilizing DNA comprising: a) isolating and purifying the DNAto produced purified DNA; b) optionally, removing metal ions from thepurified DNA; c) optionally, introducing the purified DNA into asolution to form a stabilized DNA formulation, the solution beingsubstantially free of metal ions; d) placing the purified DNA in aformulation containing an amorphous sugar; and c) lyophilizing thesolution.
 17. A stable formulation of DNA comprising demetalated DNA anda nonreducing free radical scavenging agent.
 18. The formulation ofclaim 17 which further comprises a salt.
 19. The formulation of claim 17wherein the demetalated DNA is selected from influenza virus DNA,hepatitis A virus DNA, hepatitis B virus DNA, hepatitis C virus DNA,human papillomavirus DNA, DNA from Mycobacterium tuberculosis, humanimmunodeficiency virus DNA, varicella zoster virus DNA, herpes virusDNA, measles virus DNA, rotavirus DNA, mumps virus DNA, rubella virusDNA and combinations thereof.
 20. The method of claim 1 wherein thesolution contains a metal ion chelator.
 21. The method of claim 20wherein the metal ion chelator is selected from EDTA, DTPA, NTA,inositol hexaphosphate, tripolyphosphate, polyphosphoric acid, sodiumsuccinate, potassium succinate, lithium succinate, sodium malate,potassium malate, lithium malate and combinations thereof.
 22. Astabilized DNA formulation comprising purified DNA, a nonreducing freeradical scavenging agent, a metal ion chelator, a salt and a buffer. 23.A stabilized DNA formulation of claim 22 wherein the metal ion chelatoris selected from the group consisiting of EDTA, DTPA, NTA, inositolhexaphosphate, tripolyphosphate, polyphosphoric acid, sodium succinate,potassium succinate, lithium succinate, sodium malate, potassium malate,lithium malate and combinations thereof.
 24. A stabilized DNAformulation of claim 23 wherein the nonreducing free radical scavengingagent is selected from ethanol, glycerol, methionine, dimethylsulfoxide, and combinations thereof.
 25. A stabilized DNA formulation ofclaim 24 wherein the metal ion chelator is EDTA and the nonreducing freeradical scavenging agent is ethanol.
 26. A stabilized DNA formulation ofclaim 25 wherein the salt is selected from NaCl, KCl, LiCl andcombinations thereof.
 27. A stabilized DNA formulation of claim 25wherein the salt is NaCl.
 28. A stabilized DNA formulation of claim 26wherein the NaCl concentration is from about 100 mM to 200 mM.
 28. Astabilized DNA formulation of claim 25 wherein the buffer is selectedfrom Tris-HCl, glycine, sodium phosphate, potassium phosphate, lithiumphosphate, sodium succinate, potassium succinate, lithium succinate,sodium malate, potassium malate, lithium malate, sodium bicarbonate,potassium bicarbonate, lithium bicarbonate and combinations thereof. 29.A stabilized DNA formulation of claim 28 wherein the buffer is Tris-HCl.30. A stabilized DNA formulation of claim 29 wherein the buffer pH isfrom about 9.0 to about 9.5 in a glycine buffer.
 30. A stabilized DNAformulation of claim 29 wherein the buffer pH is from about 8.0 to about9.0 in a Tris-HCl buffer.
 31. A stabilized DNA formulation of claim 25wherein EDTA is present at a concentration up to about 5 mM.
 32. Astabilized DNA formulation of claim 31 wherein EDTA is present at aconcentration up to about 500 μM.
 33. A stabilized DNA formulation ofclaim 25 wherein ethanol is present at a concentration up to about 3%.34. A stabilized DNA formulation of claim 33 wherein ethanol is presentat a concentration up to about 2%.
 35. A stabilized DNA formulationcomprising: (a) a purified DNA; (b) Tris-HCl buffer at a pH from about8.0 to about 9.0; (b) ethanol at about 3% w/v; (c) EDTA in aconcentration range up to about 5 mM; and, (d) NaCl at a concentrationfrom about 50 mM to about 500 mM.
 36. A stabilized DNA formulation ofclaim 35 wherein the NaCl concentration is from about 100 mM to 200 mM.37. A stabilized DNA formulation of claim 36 wherein the buffer pH isfrom about 8.5 to about 9.0.
 38. A stabilized DNA formulation of claim37 wherein EDTA is present at a concentration up to about 500 μM.
 39. Astabilized DNA formulation of claim 38 wherein ethanol is present at aconcentration up to about 2%.
 40. A stabilized DNA formulation of claim35 wherein EDTA is present at a concentration up to about 500 μM.
 41. Astabilized DNA formulation of claim 40 wherein ethanol is present at aconcentration up to about 2%.